Ricin-like toxin variants for treatment of cancer, viral or parasitic infections

ABSTRACT

The present invention provides a protein having an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a disease specific protease such as a cancer, fungal, viral or parasitic protease. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying mammalian cancer cells, cells infected with a virus, a fungus, or parasite, or parasites utilizing the nucleic acid molecules and proteins of the invention and pharmaceutical compositions for treating human cancer, viral infection, fungal infection, or parasitic infection.

FIELD OF THE INVENTION

The invention relates to proteins useful as therapeutics against cancer,viral infections, parasitic and fungal infections. The proteins containA and B chains of a ricin-like toxin linked by a linker sequence that isspecifically cleaved and activated by proteases specific todisease-associated pathogens or cells.

BACKGROUND OF THE INVENTION

Bacteria and plants are known to produce cytotoxic proteins which mayconsist of one, two or several polypeptides or subunits. Those proteinshaving a single subunit may be loosely classified as Type I proteins.Many of the cytotoxins which have evolved two subunit structures arereferred to as type II proteins (Saelinger, C. B. in Trafficking ofBacterial Toxins (eds. Saelinger, C. B.) 1-13 (CRC Press Inc., BocaRaton, Fla., 1990). One subunit, the A chain, possesses the toxicactivity whereas the second subunit, the B chain, binds cell surfacesand mediates entry of the toxin into a target cell. A subset of thesetoxins kill target cells by inhibiting protein biosynthesis. Forexample, bacterial toxins such as diphtheria toxin or Pseudomonasexotoxin inhibit protein synthesis by inactivating elongation factor 2.Plant toxins such as ricin, abrin, and bacterial toxin Shiga toxin,inhibit protein synthesis by directly inactivating the ribosomes(Olsnes, S. & Phil, A. in Molecular action of toxins and viruses (eds.Cohen, P. & vanHeyningen, S.) 51-105 Elsevier Biomedical Press,Amsterdam, 1982).

Ricin, derived from the seeds of Ricinus communis (castor oil plant),may be the most potent of the plant toxins. It is estimated that asingle ricin A chain is able to inactivate ribosomes at a rate of 1500ribosomes/minute. Consequently, a single molecule of ricin is enough tokill a cell (Olsnes, S. & Phil, A. in Molecular action of toxins andviruses (eds. Cohen, P. & vanHeyningen, S.) (Elsevier Biomedical Press,Amsterdam, 1982). The ricin toxin is a glycosylated heterodimerconsisting of A and B chains with molecular masses of 30,625 Da and31,431 Da linked by a disulphide bond. The A chain of ricin has anN-glycosidase activity and catalyzes the excision of a specific adenineresidue from the 28S rRNA of eukaryotic ribosomes (Endo, Y. & Tsurugi,K. J., Biol. Chem. 262:8128 (1987)). The B chain of ricin, although nottoxic in itself, promotes the toxicity of the A chain by binding togalactose residues on the surface of eukaryotic cells and stimulatingreceptor-mediated endocytosis of the toxin molecule (Simmons et al.,Biol. Chem. 261:7912 (1986)). Once the toxin molecule consisting of theA and B chains is internalized into the cell via clathrin-dependent orindependent mechanisms, the greater reduction potential within the cellinduces a release of the active A chain, eliciting its inhibitory effecton protein synthesis and its cytotoxicity (Emmanuel, F. et al., Anal.Biochem. 173: 134-141 (1988); Blum, J. S. et al., J. Biol. Chem. 266:22091-22095 (1991); Fiani, M. L. et al., Arch. Biochem. Biophys. 307:225-230 (1993)). Empirical evidence suggests that activated toxin (e.g.ricin, shiga toxin and others) in the endosomes is transcytosed throughthe trans-Golgi network to the endoplasmic reticulum by retrogradetransport before the A chain is translocated into the cytoplasm toelicit its action (Sandvig, K. & van Deurs, B., FEBS Lett. 346: 99-102(1994).

Protein toxins are initially produced in an inactive, precursor form.Ricin is initially produced as a single polypeptide (preproricin) with a35 amino acid N-terminal presequence and 12 amino acid linker betweenthe A and B chains. The pre-sequence is removed during translocation ofthe ricin precursor into the endoplasmic reticulum (Lord, J. M., Eur. J.Biochem. 146:403-409 (1985) and Lord, J. M., Eur. J. Biochem.146:411-416 (1985)). The proricin is then translocated into specializedorganelles called protein bodies where a plant protease cleaves theprotein at a linker region between the A and B chains (Lord, J. M. etal., FASAB Journal 8:201-208 (1994)). The two chains, however, remaincovalently attached by an interchain disulfide bond (cysteine 259 in theA chain to cysteine 4 in the B chain) and mature disulfide linked ricinis stored in protein bodies inside the plant cells. The A chain isinactive in proricin (O'Hare, M. et al., FEBS Lett. 273:200-204 (1990))and it is inactive in the disulfide-linked mature ricin (Richardson, P.T. et al., FEBS Lett. 255:15-20 (1989)). The ribosomes of the castorbean plant are themselves susceptible to inactivation by ricin A chain;however, as there is no cell surface galactose to permit B chainrecognition the A chain cannot re-enter the cell. The exact mechanism ofA chain release and activation in target cell cytoplasm is not known(Lord, J. M. et al., FASAB Journal 8:201-208 (1994)). However, it isknown that for activation to take place the disulfide bond between the Aand B chains must be reduced and, hence, the linkage between subunitsbroken.

Diphtheria toxin is produced by Corynebacterium diphtheriae as a 535amino acid polypeptide with a molecular weight of approximately 58kD(Greenfield, L. et al., Proc. Natl. Acad. Sci. USA 80:6853-6857 (1983);Pastan, I. et al., Annu. Rev. Biochem. 61:331-354 (1992); Collier, R. J.& Kandel, J., J. Biol. Chem. 246:1496-1503 (1971)). It is secreted as asingle-chain polypeptide consisting of 2 functional domains. Similar toproricin, the N-terminal domain (A-chain) contains the cytotoxic moietywhereas the C-terminal domain (B-chain) is responsible for binding tothe cells and facilitates toxin endocytosis. Conversely, the mechanismof cytotoxicity for diphtheria toxin is based on ADP-ribosylation ofEF-2 thereby blocking protein synthesis and producing cell death. The 2functional domains in diphtheria toxin are linked by an arginine-richpeptide sequence as well as a disulphide bond. Once the diphtheria toxinis internalized into the cell, the arginine-rich peptide linker iscleaved by trypsin-like enzymes and the disulphide bond (Cys 186-201) isreduced. The cytotoxic domain is subsequently translocated into thecytosol substantially as described above for ricin and elicits ribosomalinhibition and cytotoxicity.

Pseudomonas exotoxin is also a 66kD single-chain toxin protein secretedby Pseudomonas aeruginosa with a similar mechanism of cytotoxicity tothat of diphtheria toxin (Pastan, I. et al., Annu. Rev. Biochem.61:331-354 (1992); Ogata, M. et al., J. Biol. Chem. 267:25396-25401(1992); Vagil, M. L. et al., Infect. Immunol. 16:353-361 (1977)).Pseudomonas exotoxin consists of 3 conjoint functional domains. Thefirst domain Ia (amino acids 1-252) is responsible for cell binding andtoxin endocytosis, a second domain II (amino acids 253-364) isresponsible for toxin translocation from the endocytic vesicle to thecytosol, and a third domain III (amino acids 400-613) is responsible forprotein synthesis inhibition and cytotoxicity. After Pseudomonasexotoxin enters the cell, the liberation of the cytotoxic domain iseffected by both proteolytic cleavage of a polypeptide sequence in thesecond domain (near Arg 279) and the reduction of the disulphide bond(Cys 265-287) in the endocytic vesicles. In essence, the overall pathwayto cytotoxicity is analogous to diphtheria toxin with the exception thatthe toxin translocation domain in Pseudomonas exotoxin is structurallydistinct.

Class 2 ribosomal inhibitory proteins (RIP-2) constitute other toxinspossessing distinct functional domains for cyotoxicity and cellbinding/toxin translocation include abrin, modeccin volkensin (Sandvig,K. et al., Biochem. Soc. Trans. 21:707-711 (1993)) and mistletoe lectin(viscumin) (Olsnes, S. & Phil, A. in Molecular action of toxins andviruses (eds. Cohen, P. & van Heyningen, S.) 51-105 Elsevier BiomedicalPress, Amsterdam, 1982; and Fodstad, et al. Canc. Res. 44:862 (1984)).Some toxins such as Shiga toxin and cholera toxin also have multiplepolypeptide chains responsible for receptor binding and endocytosis.

The ricin gene has been cloned and sequenced, and the X-ray crystalstructures of the A and B chains have been described (Rutenber, E. etal. Proteins 10:240-250 (1991); Weston et al., Mol. Bio. 244:410-422,1994; Lamb and Lord, Eur. J. Biochem. 14:265 (1985); Halling, K. et al.Nucleic Acids Res. 13:8019 (1985)). Similarly, the genes for diptheriatoxin and Pseudomonas exotoxin have been cloned and sequenced, and the3-dimensional structures of the toxin proteins have been elucidated anddescribed (Columblatti, M. et al., J. Biol. Chem. 261:3030-3035 (1986);Allured, V. S. et al., Proc. Natl. Acad. Sci. USA 83:1320-1324 (1986);Gray, G.L. et al., Proc. Natl. Acad. Sci. USA 81:2645-2649 (1984);Greenfield, L. et al., Proc. Natl. Acad. Sci. USA 80:6853-6857 (1983);Collier, R. J. et al., J. Biol. Chem. 257:5283-5285 (1982)).

The potential of bacterial and plant toxins for inhibiting mammalianretroviruses, particularly acquired immunodeficiency syndrome (AIDS),has been investigated. Bacterial toxins such as Pseudomonas exotoxin-Aand subunit A of diphtheria toxin; dual chain ribosomal inhibitory planttoxins such as ricin, and single chain ribosomal inhibitory proteinssuch as trichosanthin and pokeweed antiviral protein have been used forthe elimination of HIV infected cells (Olson et al., AIDS Res. and HumanRetroviruses 7:1025-1030 (1991)). The high toxicity of these toxins formammalian cells, combined with a lack of specificity of action poses amajor problem to the development of pharmaceuticals incorporating thetoxins, such as immunotoxins.

Due to their extreme toxicity there has been much interest in makingricin-based immunotoxins as therapeutic agents for specificallydestroying or inhibiting infected or tumourous cells or tissues (Vitettaet al., Science 238:1098-1104(1987)). An immunotoxin is a conjugate of aspecific cell binding component, such as a monoclonal antibody or growthfactor and the toxin in which the two protein components are covalentlylinked. Generally, the components are chemically coupled. However, thelinkage may also be a peptide or disulfide bond. The antibody directsthe toxin to cell types presenting a specific antigen thereby providinga specificity of action not possible with the natural toxin.Immunotoxins have been made both with the entire ricin molecule (i.e.both chains) and with the ricin A chain alone (Spooner et al., Mol.Immunol. 31:117-125, (1994)).

Immunotoxins made with the ricin dimer (IT-Rs) are more potent toxinsthan those made with only the A chain (IT-As). The increased toxicity ofIT-Rs is thought to be attributed to the dual role of the B chains inbinding to the cell surface and in translocating the A chain to thecytosolic compartment of the target cell (Vitetta et al., Science238:1098-1104 (1987); Vitetta & Thorpe, Seminars in Cell Biology 2:47-58(1991)). However, the presence of the B chain in these conjugates alsopromotes the entry of the immunotoxin into nontarget cells. Even smallamounts of B chain may override the specificity of the cell-bindingcomponent as the B chain will bind nonspecifically to galactoseassociated with N-linked carbohydrates, which is present on most cells.IT-As are more specific and safer to use than IT-Rs. However, in theabsence of the B chain the A chain has greatly reduced toxicity. Due tothe reduced potency of IT-As as compared to IT-Rs, large doses of IT-Asmust be administered to patients. The large doses frequently causeimmune responses and production of neutralizing antibodies in patients(Vitetta et al., Science 238:1098-1104 (1987)). IT-As and IT-Rs bothsuffer from reduced toxicity as the A chain is not released from theconjugate into the target cell cytoplasm.

A number of immunotoxins have been designed to recognize antigens on thesurfaces of tumour cells and cells of the immune system (Pastan et al.,Annals New York Academy of Sciences 758:345-353 (1995)). A major problemwith the use of such immunotoxins is that the antibody component is itsonly targeting mechanism and the target antigen is often found onnon-target cells (Vitetta et al., Immunology Today 14:252-259 (1993)).Also, the preparation of a suitable specific cell binding component maybe problematic. For example, antigens specific for the target cell maynot be available and many potential target cells and infective organismscan alter their antigenic make up rapidly to avoid immune recognition.In view of the extreme toxicity of proteins such as ricin, the lack ofspecificity of the immunotoxins may severely limit their usefulness astherapeutics for the treatment of cancer and infectious diseases.

The insertion of intramolecular protease cleavage sites between thecytotoxic and cell-binding components of a toxin can mimic the way thatthe natural toxin is activated. European patent application no. 466,222describes the use of maize-derived pro-proteins which can be convertedinto active form by cleavage with extracellular blood enzymes such asfactor Xa, thrombin or collagenase. Garred, O. et al. (J. Biol. Chem.270:10817-10821 (1995)) documented the use of a ubiquitouscalcium-dependent serine protease, furin, to activate shiga toxin bycleavage of the trypsin-sensitive linkage between the cytotoxic A-chainand the pentamer of cell-binding B-units. Westby et al. (BioconjugateChem. 3:375-381 (1992)) documented fusion proteins which have a specificcell binding component and proricin with a protease sensitive cleavagesite specific for factor Xa within the linker sequence. O'Hare et al.(FEBS Lett. 273:200-204 (1990)) also described a recombinant fusionprotein of RTA and staphylococcal protein A joined by atrypsin-sensitive cleavage site. In view of the ubiquitous nature of theextracellular proteases utilized in these approaches, such artificialactivation of the toxin precursor or immunotoxin does not confer amechanism for intracellular toxin activation and the problems of targetspecificity and adverse immunological reactions to the cell-bindingcomponent of the immunotoxin remain.

In a variation of the approach of insertion of intramolecular proteasecleavage sites on proteins which combine a binding chain and a toxicchain, Leppla, S. H. et al. (Bacterial Protein Toxins zbl.bakt.suppl.24:431-442 (1994)) suggest the replacement of the native cleavage siteof the protective antigen (PA) produced by Bacillus anthracis with acleavage site that is recognized by cells that contain a particularprotease. PA, recognizes, binds, and thereby assists in theinternalization of lethal factor (LF) and edema toxin (ET). alsoproduced by Bacillus anthracis. However, this approach is whollydependent on the availability of LF, or ET and PA all being localized tocells wherein the modified PA can be activated by the specific protease.It does not confer a mechanism for intracellular toxin activation andpresents a problem of ensuring sufficient quantities of toxin forinternalization in target cells.

The in vitro activation of a Staphylococcus-derived pore-forming toxin,α-hemolysin by extracellular tumour-associated proteases has beendocumented (Panchel, R. G. et al., Nature Biotechnology 14:852-857(1996)). Artificial activation of α-hemolysin in vitro by said proteaseswas reported but the actual activity and utility of α-hemolysin in thedestruction of target cells were not demonstrated.

Hemolysin does not inhibit protein synthesis but is a heptamerictransmembrane pore which acts as a channel to allow leakage of moleculesup to 3 kD thereby disrupting the ionic balances of the living cell. Theα-hemolysin activation domain is likely located on the outside of thetarget cell (for activation by extracellular proteases). The triggeringmechanism in the disclosed hemolysin precursor does not involve theintracellular proteolytic cleavage of 2 functionally distinct domains.Also, the proteases used for the α-hemolysin activation areubitquitiously secreted extracellular proteases and toxin activationwould not be confined to activation in the vicinity of diseased cells.Such widespread activation of the toxin does not confer targetspecificity and limits the usefulness of said α-hemolysin toxin astherapeutics due to systemic toxicity.

A variety of proteases specifically associated with malignancy, viralinfections and parasitic infections have been identified and described.For example, cathepsin is a family of serine, cysteine or asparticendopeptidases and exopeptidases which has been implicated to play aprimary role in cancer metastasis (Schwartz, M. K., Clin. Chim. Acta237:67-78 (1995); Spiess, E. et al., J. Histochem. Cytochem. 42:917-929(1994); Scarborough, P. E. et al., Protein Sci. 2:264-276 (1993);Sloane, B. F. et al., Proc. Natl. Acad. Sci. USA 83:2483-2487 (1986);Mikkelsen, T. et al., J. Neurosurge 83:285-290 (1995)). Matrixmetalloproteinases (MMPs or matrixins) are zinc-dependent proteinasesconsisting of collagenases, matrilysin, stromelysins, gelatinases andmacrophage elastase (Krane, S. M., Ann. N.Y. Acad. Sci. 732:1-10 (1994);Woessner, J. F., Ann. N.Y. Acad. Sci. 732:11-21 (1994); Carvalho, K. etal., Biochem. Biophys. Res. Comm. 191:172-179 (1993); Nakano, A. et al.J. of Neurosurge, 83:298-307 (1995); Peng, K-W, et al. Human GeneTherapy, 8:729-738 (1997); More, D. H. et al. Gynaecologic Oncology,65:78-82 (1997)). These proteases are involved in pathological matrixremodeling. Under normal physiological conditions, regulation ofmatrixin activity is effected at the level of gene expression. Enzymaticactivity is also controlled stringently by tissue inhibitors ofmetalloproteinases (TIMPs) (Murphy, G. et al., Ann. N.Y. Acad. Sci.732:31-41 (1994)). The expression of MMP genes is reported to beactivated in inflammatory disorders (e.g. rheumatoid arthritis) andmalignancy.

In malaria, parasitic serine and aspartic proteases are involved in hosterythrocyte invasion by the Plasmodium parasite and in hemoglobincatabolism by intraerythrocytic malaria (O'Dea, K. P. et al., Mol.Biochem. Parasitol. 72:111-119 (1995); Blackman, M. J. et al., Mol.Biochem. Parasitol. 62:103-114 (1993); Cooper, J. A. et al., Mol.Biochem. Parasitol. 56:151-160 (1992); Goldberg, D. E. et al., J. Exp.Med. 173:961-969 (1991)). Schistosoma mansoni is also a pathogenicparasite which causes schistosomiasis or bilharzia. Elastinolyticproteinases have been associated specifically with the virulence of thisparticular parasite (McKerrow, J. H. et al., J. Biol. Chem.260:3703-3707 (1985)).

Welch, A. R. et al. (Proc. Natl. Acad. Sci. USA 88:107973-10800 (1991))has described a series of viral proteases which are specificallyassociated with human cytomegalovirus, human herpesviruses, Epstein-Barrvirus, varicella zoster virus-I. and infectious laryngotracheitis virus.These proteases possess similar substrate specificity and play anintegral role in viral scaffold protein restructuring in capsid assemblyand virus maturation. Other viral proteases serving similar functionshave also been documented for human T-cell leukemia virus (Blaha, I. etal., FEBS Lett. 309:389-393 (1992); Pettit, S. C. et al., J. Biol. Chem.266:14539-14547 (1991)), hepatitis viruses (Hirowatari, Y. et al., Anal.Biochem. 225:113-120 (1995); Hirowatari, Y. et al., Arch. Virol.133:349-356 (1993); Jewell, D. A. et al., Biochemistry 31:7862-7869(1992)), poliomyelitis virus (Weidner, J. R. et al., Arch. Biochem.Biophysp. 286:402-408 (1991)), and human rhinovirus (Long, A. C. et al.,FEBS Lett. 258:75-78 (1989)).

Candida yeasts are dimorphic fungi which are responsible for a majorityof opportunistic infections in AIDS patients (Holmberg, K. and Myer, R.,Scand. J. Infect. Dis. 18:179-192 (1986)). Aspartic proteinases havebeen associated specifically with numerous virulent strains of Candidaincluding Candida albican, Candida tropicalis, and Candida parapsilosis(Abad-Zapatero, C. et al., Protein Sci. 5:640-652 (1996); Cutfield, S.M. et al., Biochemistry 35:398-410 (1995); Ruchel, R. et al, Zentralbl.Bakteriol. Mikrobiol Hyg. I Abt. Orig. A. 255:537-548 (1983); Remold, H.et al., Biochim. Biophys. Acta 167:399-406 (1968)), and the levels ofthese enzymes have been correlated with the lethality of the strain(Schreiber, B, et al., Diagn. Microbiol. Infect. Dis. 3:1-5 (1985)).

SUMMARY OF THE INVENTION

The invention relates to novel recombinant toxic proteins which arespecifically toxic to diseased cells but do not depend for theirspecificity of action on a specific cell binding component. Therecombinant proteins of the invention have an A chain of a ricin-liketoxin linked to a B chain by a synthetic linker sequence which may becleaved specifically by a protease localized in cells or tissuesaffected by a specific disease to liberate the toxic A chain therebyselectively inhibiting or destroying the diseased cells or tissues. Theterm diseased cells as used herein, includes cells affected by cancer,or infected by fungi, or viruses, including retroviruses, or parasites.

Toxin targeting using the recombinant toxic proteins of the inventiontakes advantage of the fact that many DNA viruses exploit host cellulartransport mechanisms to escape immunological destruction. This isachieved by enhancing the retrograde translocation of host majorhistocompatibility complex (MHC) type I molecules from the endoplasmicreticulum into the cytoplasm (Bonifacino, J. S., Nature 384: 405-406(1996); Wiertz, E. J. et al., Nature 384: 432-438 (1996)). Thefacilitation of retrograde transport in diseased cells by the virus canenhance the transcytosis and cytotoxicity of a recombinant toxic proteinof the present invention thereby further reducing non-specificcytotoxicity and improving the overall safety of the product.

The recombinant toxic proteins of the present invention may be used totreat diseases including various forms of cancer such as T- and B-celllymphoproliferative diseases, ovarian cancer, pancreatic cancer, headand neck cancer, squamous cell carcinoma, gastrointestinal cancer,breast cancer, prostate cancer, non small cell lung cancer, malaria, anddiverse viral disease states associated with infection with humancytomegalovirus, hepatitis virus, herpes virus, human rhinovirus,infectious laryngotracheitis virus, poliomyelitis virus, or varicellazoster virus.

In one aspect, the present invention provides a purified and isolatednucleic acid having a nucleotide sequence encoding an A chain of aricin-like toxin, a B chain of a ricin-like toxin and a heterologouslinker amino acid sequence, linking the A and B chains. The linkersequence is not a native linker sequence of a ricin-like toxin, butrather a synthetic heterologous linker sequence containing a cleavagerecognition site for a disease-specific protease. The A and or the Bchain may be those of ricin.

In an embodiment, of the invention the cleavage recognition site is thecleavage recognition site for a cancer-associated protease. Inparticular embodiments, the linker amino acid sequence comprisesSLLKSRMVPNFN or SLLIARRMPNFN cleaved by cathepsin B; SKLVQASASGVN orSSYLKASDAPDN cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMNcleaved by MMP-3 (stromelysin); SLRPLALWRSFN cleaved by MMP-7(matrilysin); SPQGIAGQRNFN cleaved by MMP-9; DVDERDVRGFASFL cleaved by athermolysin-like MMP; SLPLGLWAPNFN cleaved by matrix metalloproteinase2(MMP-2); SLLIFRSWANFN cleaved by cathespin L; SGVVIATVIVIT cleaved bycathespin D; SLGPQGIWGQFN cleaved by matrix metalloproteinase 1(MMP-1);KKSPGRVVGGSV cleaved by urokinase-type plasminogen activator;PQGLLGAPGILG cleaved by membrane type 1 matrixmetalloproteinase(MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT cleaved by stromelysin 3 (orMMP-11), thermolysin, fibroblast collagenase and stromelysin-1;GPQGLAGQRGIV cleaved by matrix metalloproteinase 13 (collagenase-3);GGSGQRGRKALE cleaved by tissue-type plasminogen activator(tPA);SLSALLSSDIFN cleaved by human prostate-specific antigen; SLPRFKIIGGFNcleaved by kallikrein (bK3): SLLGIAVPGNFN cleaved by neutrophilelastase; and FFKNIVTPRTPP cleaved by calpain (calcium activated neutralprotease). The nucleic acid sequences for ricin A and B chains with eachof the linker sequences are shown in FIGS. 2D, 35C, 3D, 4D, 5D, 6D, 16D,17D, 34C, 36C, 37C, 38C, 39C, 41C, 42C, 43C, 44C, 45C, 46C and 47C,respectively.

In another embodiment, the cleavage recognition site is the cleavagerecognition site for a protease associated with the malaria parasite,Plasmondium falciparum. In particular embodiments, the linker amino acidsequence comprises QVVQLQNYDEED; LPIFGESEDNDE; QVVTGEAISVTM;ALERTFLSFPTN or KFQDMLNISQHQ. The nucleic nucleotide sequences for ricinA and B chains with each of the linker sequences are shown in FIGS. 7D,8D, 9D, 10D, and 11D.

In a another embodiment, the cleavage recognition site is the cleavagerecognition site for a viral protease. The linker sequences preferablycomprise the sequence Y-X-Y-A-Z wherein X is valine or leucine, Y is apolar amino acid, and Z is serine, asparagine or valine. In particularembodiments, the linker amino acid sequence comprises SGVVNASCRLAN orSSYVKASVSPEN cleaved by a human cytomegalovirus protease; SALVNASSAHVNor STYLQASEKFKN cleaved by a herpes simplex 1 virus protease;SSILNASVPNFN cleaved by a human herpes virus 6 protease; SQDVNAVEASSN orSVYLQASTGYGN cleaved by a varicella zoster virus protease; orSKYLQANEVITN cleaved by an infectious laryngotracheitis virus protease.The nucleic nucleotide sequences for ricin A and B chains with each ofthe linker sequences are shown in FIGS. 12D, 13D, 14D, 15D, 18D, 19D,20D, and 22D.

In another embodiment, the cleavage recognition site is the cleavagerecognition site for a hepatitis A viral protease. In particularembodiments, the linker amino acid sequence comprises SELRTQSFSNWN orSELWSQGIDDDN cleaved by a hepatitis A virus protease. The nucleicnucleotide sequences for ricin A and B chains with each of the linkersequences are shown in FIGS. 23D or 24D.

In another embodiment, the cleavage recognition site is the cleavagerecognition site for a hepatitis C viral protease. In particularembodiments, the linker amino acid sequence comprises DLEVVTSTWVFN,DEMEECASHLFN, EDVVCCSMSYFN or KGWRLLAPITAY cleaved by a hepatitis Cvirus protease. The nucleic nucleotide sequences for ricin A and Bchains with each of the linker sequences are shown in FIGS. 30C, 31C,32C and 33C.

In another embodiment, the cleavage recognition site is the cleavagerecognition site for a Candida fungal protease. In particularembodiments, the linker amino acid sequence is SKPAKFFRLNFN,SKPIEFFRLNFN or SKPAEFFALNFN cleaved by Candida aspartic protease. Thenucleic nucleotide sequences for ricin A and B chains with the firstlinker sequence are shown in FIGS. 25D.

The present invention also provides a plasmid incorporating the nucleicacid of the invention. In an embodiment, the plasmid has the restrictionmap as shown in FIGS. 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A,13A, 14A, 15A, 16A, 17A, 18A, 19A, 22A, 23A, 24A, or 25A.

In another embodiment, the present invention provides a baculovirustransfer vector incorporating the nucleic acid of the invention. Inparticular embodiments, the invention provides a baculovirus transfervector having the DNA sequence as shown in FIG. 1.

In a further embodiment, the present invention provides a baculovirustransfer vector incorporating the nucleic acid of the invention. Inparticular embodiments, the invention provides a baculovirus transfervector having the restriction map as shown in FIGS. 2C, 3C, 4C, 5C, 6C,7C, 8C, 9C, 10C, 11C, 12C, 13C, 14C, 15C, 16C, 17C, 18C, 19C, 20C, 21C,22C, 23C, 24C, 25C, 30A, 31A, 32A, 33A, 34A, 35A, 36A, 37A, 38A, 39A,40A, 41A, 42A, 43A, 44A, 45A, 46A, or 47A. or having the DNA sequence asshown in FIG. 1.

In a further aspect, the present invention provides a recombinantprotein comprising an A chain of a ricin-like toxin, a B chain of aricin-like toxin and a heterologous linker amino acid sequence, linkingthe A and B chains, wherein the linker sequence contains a cleavagerecognition site for a disease-specific protease (e.g. a cancer, viral,parasitic, or fungal protease). The A and/or the B chain may be those ofricin. In an embodiment, the cleavage recognition site is the cleavagerecognition site for a cancer, viral or parasitic protease substantiallyas described above. In a particular embodiment, the cancer is T-cell orB-cell lymphoproliferative disease. In another particular embodiment,the virus is human cytomegalovirus, Epstein-Barr virus, hepatitis virus,herpes virus, human rhinovirus, infectious laryngotracheitis virus,poliomyelitis virus, or varicella zoster virus. In a further particularembodiment, the parasite is Plasmondium falciparum.

In a further aspect, the invention provides a pharmaceutical compositionfor treating a fungal infection, such as Candida, in a mammal comprisingthe recombinant protein of the invention and a pharmaceuticallyacceptable carrier, diluent or excipient.

In yet another aspect, the invention provides a method of inhibiting ordestroying cells affected by a disease, which cells are associated witha disease specific protease, including cancer or infection with a virus,fungus, or a parasite each of which has a specific protease, comprisingthe steps of preparing a recombinant protein of the invention having aheterologous linker sequence which contains a cleavage recognition sitefor the disease-specific protease and administering the recombinantprotein to the cells. In an embodiment, the cancer is T-cell or B-celllymphoproliferative disease, ovarian cancer, pancreatic cancer, head andneck cancer, squamous cell carcinoma, gastrointestinal cancer, breastcancer, prostate cancer, non small cell lung cancer. In anotherembodiment, the virus is human cytomegalovirus, Epstein-Barr virus,hepatitis virus, herpes virus, human rhinovirus, human T-cell leukemiavirus, infectious laryngotracheitis virus, poliomyelitis virus, orvaricella zoster virus. In another embodiment, the parasite isPlasmondium falciparum.

The present invention also relates to a method of treating a mammal withdisease wherein cells affected by the disease are associated with adisease specific protease, including cancer or infection with a virus,fungus, or a parasite each of which has a specific protease byadministering an effective amount of one or more recombinant proteins ofthe invention to said mammal.

Still further, a process is provided for preparing a pharmaceutical fortreating a mammal with disease wherein cells affected by the disease areassociated with a disease specific protease, including cancer orinfection with a virus, fungus, or a parasite each of which has aspecific protease comprising the steps of preparing a purified andisolated nucleic acid having a nucleotide sequence encoding an A chainof a ricin-like toxin, a B chain of a ricin-like toxin and aheterologous linker amino acid sequence, linking the A and B chains,wherein the linker sequence contains a cleavage recognition site for thedisease-specific protease; introducing the nucleic acid into a hostcell; expressing the nucleic acid in the host cell to obtain arecombinant protein comprising an A chain of a ricin-like toxin, a Bchain of a ricin-like toxin and a heterologous linker amino acidsequence, linking the A and B chains wherein the linker sequencecontains the cleavage recognition site for the disease-specificprotease; and suspending the protein in a pharmaceutically acceptablecarrier, diluent or excipient.

In an embodiment, a process is provided for preparing a pharmaceuticalfor treating a mammal with disease wherein cells affected by the diseaseare associated with a disease specific protease, including cancer orinfection with a virus, fungus, or a parasite each of which has aspecific protease comprising the steps of identifying a cleavagerecognition site for the protease; preparing a recombinant proteincomprising an A chain of a ricin-like toxin, a B chain of a ricin-liketoxin and a heterologous linker amino acid sequence, linking the A and Bchains wherein the linker sequence contains the cleavage recognitionsite for the protease and suspending the protein in a pharmaceuticallyacceptable carrier, diluent or excipient.

In a further aspect, the invention provides a pharmaceutical compositionfor treating for treating a mammal with disease wherein cells affectedby the disease are associated with a disease specific protease,including cancer or infection with a virus, fungus, or a parasitecomprising the recombinant protein of the invention and apharmaceutically acceptable carrier, diluent or excipient.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawingsin which:

FIG. 1 shows the DNA sequence of the baculovirus transfer vector,pVL1393;

FIG. 2A summarizes the cloning strategy used to generate the pAP-213construct;

FIG. 2B shows the nucleotide sequence of the Cathepsin B linker regionsof pAP-213;

FIG. 2C shows the subcloning of the Cathepsin B linker variant into abaculovirus transfer vector;

FIG. 2D shows the DNA sequence of the pAP-214 insert containing ricinand the Cathepsin B linker;

FIG. 3A summarizes the cloning strategy used to generate the pAP-215construct;

FIG. 3B shows the nucleotide sequence of the MMP-3 linker regions ofpAP-215;

FIG. 3C shows the subcloning of the MMP-3 linker variant into abaculovirus transfer vector;

FIG. 3D shows the DNA sequence of the pAP-216 insert containing ricinand the MMP-3 linker;

FIG. 4A summarizes the cloning strategy used to generate the pAP-217construct;

FIG. 4B shows the nucleotide sequence of the MMP-7 linker regions ofpAP-217;

FIG. 4C shows the subcloning of the MMP-7 linker variant into abaculovirus transfer vector;

FIG. 4D shows the DNA sequence of the pAP-218 insert containing ricinand the MMP-7 linker;

FIG. 5A summarizes the cloning strategy used to generate the pAP-219construct;

FIG. 5B shows the nucleotide sequence of the MMP-9 linker regions ofpAP-219;

FIG. 5C shows the subcloning of the MMP-9 linker variant into abaculovirus transfer vector;

FIG. 5D shows the DNA sequence of the pAP-220 insert containing ricinand the MMP-9 linker.

FIG. 6A summarizes the cloning strategy used to generate the pAP-221construct;

FIG. 6B shows the nucleotide sequence of the thermolysin-like MMP linkerregions of pAP-221;

FIG. 6C shows the subcloning of the thermolysin-like MMP linker variantinto a baculovirus transfer vector.

FIG. 6D shows the DNA sequence of the pAP-222 insert containing ricinand the thermolysin-like MMP linker;

FIG. 7A summarizes the cloning strategy used to generate the pAP-223construct;

FIG. 7B shows the nucleotide sequence of the Plasmondium falciparum-Alinker regions of pAP-223;

FIG. 7C shows the subcloning of the Plasmondium falciparum-A linkervariant into a baculovirus transfer vector;

FIG. 7D shows the DNA sequence of the pAP-224 insert containing ricinand the Plasmondium faciparum-A linker;

FIG. 8A summarizes the cloning strategy used to generate the pAP-225construct;

FIG. 8B shows the nucleotide sequence of the Plasmondium faciparum-Blinker regions of pAP-225;

FIG. 8C shows the subcloning of the Plasmondium falciparum-B linkervariant into a baculovirus transfer vector;

FIG. 8D shows the DNA sequence of the pAP-226 insert containing ricinand the Plasmondium faciparum-B linker;

FIG. 9A summarizes the cloning strategy used to generate the pAP-227construct;

FIG. 9B shows the nucleotide sequence of the Plasmondium faciparum-Clinker regions of pAP-227;

FIG. 9C shows the subcloning of the Plasmondium falciparum-C linkervariant into a baculovirus transfer vector;

FIG. 9D shows the DNA sequence of the pAP-228 insert containing ricinand the Plasmondium faciparum-C linker;

FIG. 10A summarizes the cloning strategy used to generate the pAP-229construct;

FIG. 10B shows the nucleotide sequence of the Plasmondium faciparum-Dlinker regions of pAP-229;

FIG. 10C shows the subcloning of the Plasmondium falciparum-D linkervariant into a baculovirus transfer vector;

FIG. 10D shows the DNA sequence of the pAP-230 insert containing ricinand the Plasmondium faciparum-D linker;

FIG. 11A summarizes the cloning strategy used to generate the pAP-231construct;

FIG. 11B shows the nucleotide sequence of the Plasmondium faciparum-Elinker regions of pAP-231;

FIG. 11C shows the subcloning of the Plasmondium falciparum-E linkervariant into a baculovirus transfer vector;

FIG. 11D shows the DNA sequence of the pAP-232 insert containing ricinand the Plasmondium faciparum-E linker;

FIG. 12A summarizes the cloning strategy used to generate the pAP-233construct;

FIG. 12B shows the nucleotide sequence of the HSV-A inker regions ofpAP-233;

FIG. 12C shows the subcloning of the HSV-A linker variant into abaculovirus transfer vector;

FIG. 12D shows the DNA sequence of the pAP-234 insert containing ricinand the HSV-A linker;

FIG. 13A summarizes the cloning strategy used to generate the pAP-235construct;

FIG. 13B shows the nucleotide sequence of the HSV-B linker regions ofpAP-235;

FIG. 13C shows the subcloning of the HSV-B linker variant into abaculovirus transfer vector;

FIG. 13D shows the DNA sequence of the pAP-236 insert containing ricinand the HSV-B linker;

FIG. 14A summarizes the cloning strategy used to generate the pAP-237construct;

FIG. 14B shows the nucleotide sequence of the VZV-A linker regions ofpAP-237;

FIG. 14C shows the subcloning of the VZV-A linker variant into abaculovirus transfer vector;

FIG. 14D shows the DNA sequence of the pAP-238 insert containing ricinand the VZV-A linker;

FIG. 15A summarizes the cloning strategy used to generate the pAP-239construct;

FIG. 15B shows the nucleotide sequence of the VZV-B linker regions ofpAP-239;

FIG. 15C shows the subcloning of the VZV-B linker variant into abaculovirus transfer vector;

FIG. 15D shows the DNA sequence of the pAP-240 insert containing ricinand the VZV-B linker;

FIG. 16A summarizes the cloning strategy used to generate the pAP-241construct;

FIG. 16B shows the nucleotide sequence of the EBV-A linker regions ofpAP-241;

FIG. 16C shows the subcloning of the EBV-A linker variant into abaculovirus transfer vector;

FIG. 16D shows the DNA sequence of the pAP-242 insert containing ricinand the EBV-A linker;

FIG. 17A summarizes the cloning strategy used to generate the pAP-243construct;

FIG. 17B shows the nucleotide sequence of the EBV-B linker regions ofpAP-243;

FIG. 17C shows the subcloning of the EBV-B linker variant into abaculovirus transfer vector;

FIG. 17D shows the DNA sequence of the pAP-244 insert containing ricinand the EBV-B linker;

FIG. 18A summarizes the cloning strategy used to generate the pAP-245construct;

FIG. 18B shows the nucleotide sequence of the CMV-A linker regions ofpAP-245;

FIG. 18C shows the subcloning of the CMV-A linker variant into abaculovirus transfer vector;

FIG. 18D shows the DNA sequence of the pAP-246 insert containing ricinand the CMV-A linker;

FIG. 19A summarizes the cloning strategy used to generate the pAP-247construct;

FIG. 19B shows the nucleotide sequence of the CMV-B linker regions ofpAP-247;

FIG. 19C shows the subcloning of the CMV-B linker variant into abaculovirus transfer vector;

FIG. 19D shows the DNA sequence of the pAP-248 insert containing ricinand the CMV-B linker.

FIG. 20A summarizes the cloning strategy used to generate the pAP-249construct;

FIG. 20B shows the nucleotide sequence of the HHV-6 linker regions ofpAP-249;

FIG. 20C shows the subcloning of the HHV-6 linker variant into abaculovirus transfer vector;

FIG. 20D shows the DNA sequence of the pAP-250 insert containing ricinand the HHV-6 linker;

FIG. 21 shows the amino acid sequences of the wild type ricin linker andcancer protease-sensitive amino acid linkers contained in pAP-213 topAP-222 and linkers pAP-241 to pAP-244;

FIG. 22A summarizes the cloning strategy used to generate the pAP-253construct;

FIG. 22B shows the nucleotide sequence of the ILV linker regions ofpAP-253;

FIG. 22C shows the subcloning of the ILV linker variant into abaculovirus transfer vector;

FIG. 22D shows the DNA sequence of the pAP-254 insert containing ricinand the ILV linker;

FIG. 23A summarizes the cloning strategy used to generate the pAP-257construct;

FIG. 23B shows the nucleotide sequence of the HAV-A linker regions ofpAP-257;

FIG. 23C shows the subcloning of the HAV-A linker variant into abaculovirus transfer vector;

FIG. 23D shows the DNA sequence of the pAP-258 insert containing ricinand the HAV-A linker;

FIG. 24A summarizes the cloning strategy used to generate the pAP-255construct;

FIG. 24B shows the nucleotide sequence of the HAV-B linker regions ofpAP-255;

FIG. 24C shows the subcloning of the HAV-B linker ariant into abaculovirus transfer vector;

FIG. 24D shows the DNA sequence of the pAP-256 nsert containing ricinand the HAV-B linker;

FIG. 25A summarizes the cloning strategy used to generate the pAP-259construct;

FIG. 25B shows the nucleotide sequence of the CAN linker regions ofpAP-259;

FIG. 25C shows the subcloning of the CAN linker variant into abaculovirus transfer vector;

FIG. 25D shows the DNA sequence of the pAP-260 insert containing ricinand the CAN linker;

FIG. 26 shows the amino acid sequences of the wild type ricin linker andPlasmondium falciparum protease-sensitive amino acid linkers containedin linkers pAP-223 to pAP-232;

FIG. 27 shows the amino acid sequences of the wild type ricin linker andthe viral protease-sensitive amino acid linkers contained in pAP-233 topAP-240, pAP-245-pAP-248, pAP-253 to pAP-258;

FIG. 28 shows the amino acid sequences of the wild type ricin linker andthe Candida aspartic protease-sensitive amino acid linker contained inpAP-259 to pAP-264;

FIG. 29 describes an alternative mutagenesis and subcloning strategy toprovide a baculovirus transfer vector containing the ricin-like toxinvariant gene; and

FIG. 30A summarizes the cloning strategy used to generate the pAP-262construct;

FIG. 30B shows the nucleotide sequence of the HCV-A linker region ofpAP-262;

FIG. 30C shows the DNA sequence of the pAP-262 insert;

FIG. 30D shows the amino acid sequence comparison of utant preproricinlinker region HCV-A to wild type;

FIG. 31A summarizes the cloning strategy used to enerate the pAP-264construct;

FIG. 31B shows the nucleotide sequence of the HCV-B linker region ofpAP-264;

FIG. 31C shows the DNA sequence of the pAP-264 insert;

FIG. 31D shows the amino acid sequence comparison of mutant preproricinlinker region HCV-B to wild type;

FIG. 32A summarizes the cloning strategy used to generate the pAP-266construct;

FIG. 32B shows the nucleotide sequence of the HCV-C linker region ofpAP-266;

FIG. 32C shows the DNA sequence of the pAP-266 insert;

FIG. 32D shows the amino acid sequence comparison of mutant preproricinlinker region HCV-C to wild type;

FIG. 33A summarizes the cloning strategy used to generate the pAP-268construct;

FIG. 33B shows the nucleotide sequence of the HCV-D linker region ofpAP-268;

FIG. 33C shows the DNA sequence of the pAP-268 insert;

FIG. 33D shows the amino acid sequence comparison of mutant preproricinlinker region HCV-D to wild type;

FIG. 34A summarizes the cloning strategy used to generate the pAP-270construct;

FIG. 34B shows the nucleotide sequence of the MMP-2 linker region ofpAP-270;

FIG. 34C shows the DNA sequence of the pAP-270 insert;

FIG. 34D shows the amino acid sequence comparison of mutant preproricinlinker region of MMP-2 to wild type;

FIG. 35A summarizes the cloning strategy used to generate the pAP-272construct;

FIG. 35B shows the nucleotide sequence of the Cathepsin B (Site 2)linker region of pAP-272;

FIG. 35C shows the DNA sequence of the pAP-272 insert;

FIG. 35D shows the amino acid sequence comparison of mutant preproricinlinker region of Cathepsin B (Site 2) to wild type;

FIG. 36A summarizes the cloning strategy used to generate the pAP-274construct;

FIG. 36B shows the nucleotide sequence of the Cathepsin L linker regionof pAP-274;

FIG. 36C shows the DNA sequence of the pAP-274 insert;

FIG. 36D shows the amino acid sequence comparison of mutant preproricinlinker region of Cathepsin L to wild type;

FIG. 37A summarizes the cloning strategy used to generate the pAP-276construct;

FIG. 37B shows the nucleotide sequence of the Cathepsin D linker regionof pAP-276;

FIG. 37C shows the DNA sequence of the pAP-276 insert;

FIG. 37D shows the amino acid sequence comparison of mutant preproricinlinker region of Cathepsin D to wild type;

FIG. 38A summarizes the cloning strategy used to generate the pAP-278construct;

FIG. 38B shows the nucleotide sequence of the MMP-1 linker region ofpAP-278;

FIG. 38C shows the DNA sequence of the pAP-278 insert;

FIG. 38D shows the amino acid sequence comparison of mutant preproricinlinker region of MMP-1 to wild type;

FIG. 39A summarizes the cloning strategy used to generate the pAP-280construct;

FIG. 39B shows the nucleotide sequence of the Urokinase-Type PlasminogenActivator linker region of pAP-280;

FIG. 39C shows the DNA sequence of the pAP-280 insert;

FIG. 39D shows the amino acid sequence comparison of mutant preproricinlinker region of Urokinase-Type Plasminogen Activator to wild type;

FIG. 40A summarizes the cloning strategy used to generate the pAP-282construct;

FIG. 40B shows the nucleotide sequence of the MT-MMP linker region ofpAP-282;

FIG. 40C shows the DNA sequence of the pAP-282 insert;

FIG. 40D shows the amino acid sequence comparison of mutant preproricinlinker region of MT-MMP to wild type;

FIG. 41A summarizes the cloning strategy used to generate the pAP-284construct;

FIG. 41B shows the nucleotide sequence of the MMP-11 linker region ofpAP-284;

FIG. 41C shows the DNA sequence of the pAP-284 insert;

FIG. 41D shows the amino acid sequence comparison of mutant preproricinlinker region of MMP-11 to wild type;

FIG. 42A summarizes the cloning strategy used to generate the pAP-286construct;

FIG. 42B shows the nucleotide sequence of the MMP-13 linker region ofpAP-286;

FIG. 42C shows the DNA sequence of the pAP-286 insert;

FIG. 42D shows the amino acid sequence comparison of utant preproricinlinker region of MMP-13 to wild type;

FIG. 43A summarizes the cloning strategy used to generate the pAP-288construct;

FIG. 43B shows the nucleotide sequence of the Tissue-type PlasminogenActivator linker region of pAP-288;

FIG. 43C shows the DNA sequence of the pAP-288 insert;

FIG. 43D shows the amino acid sequence comparison of mutant preproricinlinker region of Tissue-type Plasminogen Activator to wild type;

FIG. 44A summarizes the cloning strategy used to generate the pAP-290construct;

FIG. 44B shows the nucleotide sequence of the human Prostate-SpecificAntigen linker region of pAP-290;

FIG. 44C shows the DNA sequence of the pAP-290 insert;

FIG. 44D shows the amino acid sequence comparison of mutant preproricinlinker region of the human Prostate-Specific Antigen to wild type;

FIG. 45A summarizes the cloning strategy used to generate the pAP-292construct;

FIG. 45B shows the nucleotide sequence of the kallikrein linker regionof pAP-292;

FIG. 45C shows the DNA sequence of the pAP-292 insert;

FIG. 45D shows the amino acid sequence comparison of mutant preproricinlinker region of the kallikrein to wild type;

FIG. 46A summarizes the cloning strategy used to generate the pAP-294construct;

FIG. 46B shows the nucleotide sequence of the neutrophil elastase linkerregion of pAP-294;

FIG. 46C shows the DNA sequence of the pAP-294 insert;

FIG. 46D shows the amino acid sequence comparison of mutant preproricinlinker region of neutrophil elastase to wild type;

FIG. 47A summarizes the cloning strategy used to generate the pAP-296construct;

FIG. 47B shows the nucleotide sequence of the calpain linker region ofpAP-296;

FIG. 47C shows the DNA sequence of the pAP-296 insert;

FIG. 47D shows the amino acid sequence comparison of mutant preproricinlinker region of calpain to wild type;

FIG. 48 is a blot showing cleavage of pAP-214 by Cathepsin B;

FIG. 49 is a blot showing cleavage of pAP-220 with MMP-9;

FIG. 50 is a blot showing activation of pAP-214; and

FIG. 51 is a blot showing activation of pAP-220.

FIG. 52 is a blot showing cleavage of pAP-248 with HCMV.

FIG. 53 is a blot showing activation of pAP-248.

FIG. 54 is a blot showing cleavage of pAP-256 by HAV 3C.

FIG. 55 is a blot showing activation of pAP-256.

FIG. 56 is a semi-logithmic graph illustrating the cytotoxicity to COS-1cells of undigested pAP-214 and pAP-214 digestedwith Cathepsin B.

FIG. 57 is a semi-logithmic graph illustrating the cytotoxicity ofpAP-220 digested with MMP-9 compared to freshly thawed pAP-220 and ricinon COS1 cells.

FIG. 58 is a blot showing cleavage of pAP-270 with MMP-2.

FIG. 59 is a blot showing activation of pAP-270.

FIG. 60 is a blot showing cleavage of pAP-288 by t-PA.

FIG. 61 is a blot showing activation of pAP-288.

FIG. 62 is a blot showing cleavage of pAP-294 with human neutrophilelastase.

FIG. 63 is a blot showing activation of pAP-294.

FIG. 64 is a blot showing cleavage of pAP-296 with calpain.

FIG. 65 is a blot showing activation of pAP-296.

FIG. 66 is a blot showing cleavage of pAP-222 with MMP-2.

FIG. 67 is a blot showing activation of pAP-222.

DETAILED DESCRIPTION OF THE INVENTION Nucleic Acid Molecules of theInvention

As mentioned above, the present invention relates to novel nucleic acidmolecules comprising a nucleotide sequence encoding an A chain of aricin-like toxin, a B chain of a ricin- like toxin and a heterologouslinker amino acid sequence, linking the A and B chains. The heterologouslinker sequence contains a cleavage recognition site for adisease-specific protease (e.g. a viral protease, parasitic protease,cancer-associated protease, or a fungal protease).

The term “isolated and purified” as used herein refers to a nucleic acidsubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An “isolated and purified”nucleic acid is also substantially free of sequences which naturallyflank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends ofthe nucleic acid) from which the nucleic acid is derived. The term“nucleic acid” is intended to include DNA and RNA and can be eitherdouble stranded or single stranded.

The term “linker sequence” as used herein refers to an internal aminoacid sequence within the protein encoded by the nucleic acid molecule ofthe invention which contains residues linking the A and B chain so as torender the A chain incapable of exerting its toxic effect, for examplecatalytically inhibiting translation of a eukaryotic ribosome. Byheterologous is meant that the linker sequence is not a sequence nativeto the A or B chain of a ricin-like toxin or precursor thereof. However,preferably, the linker sequence may be of a similar length to the linkersequence of a ricin-like toxin and should not interfere with the role ofthe B chain in cell binding and transport into the cytoplasm. When thelinker sequence is cleaved the A chain becomes active or toxic.

The nucleic acid molecule of the invention is cloned by subjecting apreproricin cDNA clone to site-directed mutagenesis in order to generatea series of variants differing only in the sequence between the A and Bchains (linker region). Oligonucleotides, corresponding to the extreme5′ and 3′ ends of the preproricin gene are synthesized and used to PCRamplify the gene. Using the cDNA sequence for preproricin (Lamb et al.,Eur. J. Biochem. 145:266-270 (1985)), several oligonucleotide primersare designed to flank the start and stop codons of the preproricin openreading frame.

The preproricin cDNA is amplified using the upstream primer Ricin-99 orRicin-109 and the downstream primer Ricin1729C with Vent DNA polymerase(New England Biolabs) using standard procedures (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor Laboratory Press, 1989)). The purified PCR fragment encoding thepreproricin cDNA is then ligated into an Eco RI-digested pbluescript IISK plasmid (Stratagene), and is used to transform competent XL1-Bluecells (Stratagene). The cloned PCR product containing the putativepreproricin gene is confirmed by DNA sequencing of the entire cDNA clone. The sequences and location of oligonucleotide primers used forsequencing are shown in Table 1.

The preproricin cDNA clone is subjected to site directed mutagenesis inorder to generate a series of variants differing only in the sequencebetween the A and B chains (linker region). The wild-type preproricinlinker region is replaced with the heterogenous linker sequences thatare cleaved by the various disease-specific proteases as shown in FIGS.21, 26, 27, 28, and Part D of FIGS. 30-47. Linker identification as usedherein in connection with the sequences provided in these figures havebeen assigned the sequence ID numbers as discussed below.

The linker regions of the variants encode a cleavage recognitionsequence for a disease-specific protease associated with for example,cancer, viruses, parasites, or fungii. The mutagenesis and cloningstrategy used to generate the disease-specific protease-sensitive linkervariants are summarized in Part A of FIGS. 2-20, and Part A of FIGS.22-25. The first step involves a DNA amplification using a set ofmutagenic primers in combination with the two flanking primersRichin-99Eco or Ricin-109Eco and Ricin1729C Pst I. Restriction digestedPCR fragments are gel purified and then ligated with PBluescript SKwhich has been digested with Eco RI and Pst I. Ligation reactions areused to transform competent XL1-Blue cells (Stratagene). Recombinantclones are identified by restriction digests of plasmid miniprep DNA andthe mutant linker sequences are confirmed by DNA sequencing. Withrespect to the nucleotide sequences and amino acid sequences prepared asa result of the implementation of this strategy the following sequenceshave been assigned the sequence ID numbers as indicated.

SEQ ID NO. 1 is used herein in connection with the DNA sequence of thebaculovirus transfer vector, pVL1393.

The nucleotide sequence of Cathepsin B linker regions of pAP-213 arereferred to herein as SEQ ID NO. 2.

The nucleotide sequence of Cathepsin B linker regions of pAP-214 arereferred to herein as SEQ ID NO. 3.

The nucleotide sequence of MMP-3 linker regions of pAP-215 are referredto herein as SEQ ID NO. 4.

The DNA sequence of the pAP-216 insert containing ricin and the MMP-3linker are referred to herein as SEQ ID NO. 5.

The nucleotide sequence of MMP-7 linker regions of pAP-217 are referredto herein as SEQ ID NO. 6.

The DNA sequence of the pAP-218 insert containing ricin and the MMP-7linker are referred to herein as SEQ ID NO. 7.

The nucleotide sequence of MMP-9 linker regions of pAP-219 are referredto herein as SEQ ID NO. 8.

The DNA sequence of the pAP-220 insert containing ricin and the MMP-9are referred to herein as SEQ ID NO. 9.

The nucleotide sequence of thermolysin-like MMP linker regions ofpAP-221 are referred to herein as SEQ ID NO. 10.

The DNA sequence of of pAP-222 insert containing ricin and thethermolysin-like MMP linker are referred to herein as SEQ ID NO. 11.

The nucleotide sequence of Plasmondium faciparum-A linker regions ofpAP-223 are referred to herein as SEQ ID NO. 12.

The DNA sequence of the pAP-224 insert containing ricin and thePlasmondium faciparum-A linker are referred to herein as SEQ ID NO. 13.

The nucleotide sequence of Plasmondium faciparum-B linker regions ofpAP-225 are referred to herein as SEQ ID NO. 14.

The DNA sequence of Plas the pAP-226 insert containing ricin and thePlasmondium faciparum-B linker are referred to herein as SEQ ID NO. 15.

The nucleotide sequence of Plasmondium faciparum-C linker regions ofpAP-227 are referred to herein as SEQ ID NO. 16.

The DNA sequence of the pAP-228 insert containing ricin and thePlasmondium faciparum-C linker are referred to herein as SEQ ID NO. 17.

The nucleotide sequence of the the Plasmodium falciparum-D linkerregions of pAP-229 is referred to herein as SEQ ID NO. 18.

The DNA sequence of the pAP-230 insert containing ricin and thePlasmondium faciparum-D linker is referred to herein as SEQ ID NO. 19.

The nucleotide sequence of the Plasmondium falciparum-E linker regionsof pAP-231 is referred to herein as SEQ ID NO. 20.

The DNA sequence of the pAP-232 insert containing ricin and thePlasmondium faciparum-E linker is referred to herein as SEQ ID NO. 21.

The nucleotide sequence of the HSV-A linker regions of pAP-233 isreferred to herein as SEQ ID NO. 22.

The DNA sequence of the pAP-234 insert containing ricin and the HSV-Alinker is referred to herein as SEQ ID NO. 23.

The nucleotide sequence of the HSV-B linker regions of pAP-235 isreferred to herein as SEQ ID NO. 24.

The DNA sequence of the pAP-236 insert containing ricin and the HSV-Blinker is referred to herein as SEQ ID NO. 25.

The nucleotide sequence of the VZV-A linker regions of pAP-237 arereferred to herein as SEQ ID NO. 26.

The DNA sequence of the pAP-238 insert containing ricin and the VZV-Alinker are referred to herein as SEQ ID NO. 27.

The nucleotide sequence of the VZV-B linker regions of PAP-239 isreferred to herein as SEQ ID NO. 28.

The DNA sequence of the pAP-240 insert containing ricin and the VZV-Blinker is referred to herein as SEQ ID NO. 29.

The nucleotide sequence of the EBV-A linker regions of pAP-241 isreferred to herein as SEQ ID NO. 30.

The DNA sequence of the pAP-242 insert containing ricin and the EBV-Alinker is referred to herein as SEQ ID NO. 31.

The nucleotide sequence of the EBV-B linker regions of pAP-243 isreferred to herein as SEQ ID NO. 32.

The DNA sequence of the pAP-244 insert containing ricin and the EBV-Blinker is referred to herein as SEQ ID NO. 33.

The nucleotide sequence of the CMV-A linker regions of pAP-245 isreferred to herein as SEQ ID NO. 34.

The DNA sequence of the pAP-246 insert containing ricin and the CMV-Alinker is referred to herein as SEQ ID NO. 35.

The nucleotide sequence of the CMV-B linker regions of pAP-247 isreferred to herein as SEQ ID NO. 36.

The DNA sequence of the pAP-248 insert containing ricin and the CMV-Blinker is referred to herein as SEQ ID NO. 37.

The nucleotide sequence of the HHV-6 linker regions of pAP-249 isreferred to herein as SEQ ID NO. 38.

The DNA sequence of the pAP-250 insert containing ricin and the HHV-6linker is referred to herein as SEQ ID NO. 39.

The amino acid sequences of the cancer protease-sensitive amino acidlinkers contained in the following pAP proteins have the sequence IDnumbers as indicated: pAP-213 and pAP-214 (SEQ ID NO. 40); pAP-215 andpAP-216 (SEQ ID NO. 41); pAP-217 and pAP-218; (SEQ ID NO. 42); pAP-219and pAP-220 (SEQ ID NO. 43); and pAP-221 and pAP-222 (SEQ ID NO. 44).

The amino acid sequences of the following cancer protease-sensitivelinkers are referred to herein with the corresponding sequence IDnumbers: pAP-241 and pAP-242 (SEQ ID NO. 45); and pAP-243 and pAP-244(SEQ ID NO. 46).

The nucleotide sequence of the ILV linker regions of pAP-253 is referredto herein as SEQ ID NO. 47.

The DNA sequence of the pAP-254 insert containing ricin and the ILVlinker is referred to herein as SEQ ID NO. 48.

The nucleotide sequence of the HAV-A linker regions of pAP-257 isreferred to herein as SEQ ID NO. 49.

The DNA sequence of the pAP-258 insert containing ricin and HAV-A linkeris referred to herein as SEQ ID NO. 50.

The nucleotide sequence of the HAV-B linker regions of pAP-255 isreferred to herein as SEQ ID NO. 51.

The DNA sequence of the pAP-256 insert containing ricin and the HAV-Blinker is referred to herein as SEQ ID NO. 52.

The nucleotide sequence of the CAN linker regions of pAP-259 is referredto herein as SEQ ID NO. 53.

The DNA sequence of the pAP-260 insert containing ricin and the CANlinker is referred to herein as SEQ ID NO. 54.

The amino acid sequences of Plasmondium falciparum protease-sensitivelinkers are referred to herein by the sequence ID numbers as follows:pAP-223 and pAP-224 (SEQ ID NO 55); pAP-225 and pAP-226 (SEQ ID NO 56);pAP-227 and pAP-228 (SEQ ID NO 57); pAP-229 and pAP-230 (SEQ ID NO 58);and pAP-231 and pAP-232 (SEQ ID NO 59) (see FIG. 26).

The amino acid sequences of the viral protease-sensitive linkers whichfollow are referred to herein by the sequence ID numbers indicated:pAP-233 and pAP 234 (SEQ ID NO 60); pAP-235 and pAP-236 (SEQ ID NO 61);and pAP-249 and pAP-250 (SEQ ID NO 62) (see FIG. 27).

The amino acid sequences of the viral protease-sensitive linkers whichfollow are referred to herein by the sequence ID numbers indicated:pAP-245 and pAP-246 (SEQ ID NO 63); and pAP-247 and pAP-248 (SEQ ID NO64) (see FIG. 27).

The amino acid sequences of the viral protease-sensitive linkers whichfollow are referred to herein by the sequence ID numbers indicated:pAP-237 and pAP-238 (SEQ ID NO 65); and pAP-239 and pAP-240 (SEQ ID NO66); pAP-253 and pAP-254 (SEQ ID NO 67); pAP-255 and pAP-256 (SEQ ID NO68); and pAP-257 and pAP-258 (SEQ ID NO 69) (see FIG. 27).

The amino acid sequences of the Candida aspartic protease-sensitivelinkers are referred to herein by the sequence ID numbers indicated:pAP-259 and pAP-260 (SEQ ID NO 70); pAP-261 and pAP-262 (SEQ ID NO 71);and pAP-263 and pAP-264 (SEQ ID NO 72).

An alternative mutagenesis and cloning strategy that can be used togenerate the disease-specific protease-sensitive linker variants issummarized in FIG. 29. The first step of this method involves a DNAamplification using a set of mutagenic primers in combination with thetwo flanking primers Ricin-109Eco and Ricin1729Pst. Restriction digestedPCR fragments (Eco RI and Pst I) are gel purified. Preproricin variantsproduced from this method can be subcloned directly into the baculovirustransfer vector digested with Eco RI and Pst I and intermediate ligationsteps involving pBluescript SK and pSB2 are circumvented. The cloningstrategies used to generate disease-specific protease-sensitive linkervariants are summarized in Part A of FIGS. 30 to 47. With respect to thenucleotide sequences and amino acid sequences prepared as a result ofthe implementation of this strategy the following sequences have beenassigned the sequence ID numbers as indicated.

The nucleotide sequence of the HCV-A linker region of pAP-262 isreferred to herein as SEQ ID NO. 73.

The DNA sequence of the pAP-262 insert is referred to herein as SEQ IDNO. 74.

The amino acid sequence of the mutant preproricin linker region forHCV-A, pAP-262, is referred to herein as SEQ ID NO. 75.

The nucleotide sequence of the HCV-B linker region of pAP-264 isreferred to herein as SEQ ID NO. 76.

The DNA sequence of the pAP-264 insert is referred to herein as SEQ IDNO. 77.

The amino acid sequence of the mutant preproricin linker region forHCV-B, pAP-264, is referred to herein as SEQ ID NO. 8.

The nucleotide sequence of the HCV-C linker region of AP-266 is referredto herein as SEQ ID NO. 79.

The DNA sequence of the pAP-266 insert is referred to herein as SEQ IDNO. 80.

The amino acid sequence of the mutant preproricin linker region forHCV-C, pAP-266, is referred to herein as SEQ ID NO. 81.

The nucleotide sequence of the HCV-D linker region of pAP-268 isreferred to herein as SEQ ID NO. 82.

The DNA sequence of the pAP-268 insert is referred to herein as SEQ IDNO. 83.

The amino acid sequence of the mutant preproricin linker region forHCV-D, pAP-268, is referred to herein as SEQ ID NO. 84.

The nucleotide sequence of the MMP-2 linker region of pAP-270 isreferred to herein as SEQ ID NO. 85.

The DNA sequence of the pAP-270 insert is referred to herein as SEQ IDNO. 86.

The amino acid sequence of the mutant preproricin linker region forMMP-2, pAP-270, is referred to herein as SEQ ID NO. 87.

The nucleotide acid sequence of the Cathepsin B (Site 2) linker regionof pAP-272 is referred to herein as SEQ ID NO. 88.

The DNA sequence of the pAP-272 insert is referred to herein as SEQ IDNO. 89.

The amino acid sequence of the mutant preproricin linker region forCathepsin B (Site 2), pAP-272, is referred to herein as SEQ ID NO. 90.

The nucleotide sequence of the Cathepsin L linker region of pAP-274 isreferred to herein as SEQ ID NO. 91.

The DNA sequence of the pAP-274 insert is referred to herein as SEQ IDNO. 92.

The amino acid sequence of the mutant preproricin linker region ofCathepsin L, pAP-274, is referred to herein as SEQ ID NO. 93.

The nucleotide sequence of Cathepsin D linker region of pAP-276 isreferred to herein as SEQ ID NO. 94.

The DNA sequence of the pAP-276 insert is referred to herein as SEQ IDNO. 95.

The amino acid sequence of the mutant preproricin linker region forCathepsin D, pAP-276, is referred to herein as SEQ ID NO. 96.

The nucleotide sequence of the MMP-1 linker region of pAP-278 isreferred to herein as SEQ ID NO. 97.

The DNA sequence of the pAP-278 insert is referred to herein as SEQ IDNO. 98.

The amino acid sequence of the mutant preproricin linker region forMMP-1, pAP-278, is referred to herein as SEQ ID NO. 99.

The nucleotide sequence of the Urokinase-Type Plasminogen Activatorlinker region of pAP-280 is referred to herein as SEQ ID NO. 100.

The DNA sequene of the pAP-280 insert is referred to herein as SEQ IDNO. 101.

The amino acid sequence of the mutant preproricin linker region forUrokinase-Type Plasminogen Activator, pAP-280, is referred to herein asSEQ ID NO. 102.

The nucleotide sequence of MT-MMP linker region of pAP-282 is referredto herein as SEQ ID NO. 103.

The DNA sequence of the pAP-282 insert is referred to herein as SEQ IDNO. 104.

The amino acid sequence of the mutant preproricin linker region forMT-MMP, pAP-282, is referred to herein as SEQ ID NO. 105.

The nucleotide sequence of the MMP-11 linker region of pAP-284 isreferred to herein as SEQ ID NO. 106.

The DNA sequence of the pAP-284 insert is referred to herein as SEQ IDNO. 107.

The amino acid sequence of the mutant preproricin linker region forMMP-11, pAP-284, is referred to herein as SEQ ID NO. 108.

The nucleotide sequence of the MMP-13 linker region of pAP-286 isreferred to herein as SEQ ID NO. 109.

The DNA sequence of the pAP-286 insert is referred to herein as SEQ IDNO. 110.

The amino acid sequence of the mutant preproricin linker region forMMP-13, pAP-286, is referred to herein as SEQ ID NO. 111.

The nucleotide sequence of the Tissue-type Plasminogen Activator linkerregion of pAP-288 is referred to herein as SEQ ID NO. 112.

The DNA sequence of the pAP-288 insert is referred to herein as SEQ IDNO. 113.

The amino acid sequence of the mutant preproricin linker region forTissue-type Plasminogen Activator, pAP-288, is referred to herein as SEQID NO. 114.

The nucleotide sequence of the human Prostate-Specific Antigen linkerregion of pAP-290 is referred to herein as SEQ ID NO. 115.

The DNA sequence of the pAP-290 insert is referred to herein as SEQ IDNO. 116.

The amino acid sequence of the mutant preproricin linker region for thehuman Prostate-Specific Antigen, pAP-290, is referred to herein as SEQID NO. 117.

The nucleotide sequence of the kallikrein linker region of pAP-292 isreferred to herein as SEQ ID NO. 118.

The DNA sequence of the pAP-292 insert is referred to herein as SEQ IDNO. 119.

The amino acid sequence of the mutant preproricin linker region for thekallikrein, pAP-292, is referred to herein as SEQ ID NO. 120.

The nucleotide sequence of the neutrophil elastase linker region ofpAP-294 is referred to herein as SEQ ID NO. 121.

The DNA sequence of the pAP-294 insert is referred to herein as SEQ IDNO. 122.

The amino acid sequence of the mutant preproricin linker region forneutrophil elastase, pAP-294, is referred to herein as SEQ ID NO. 123.

The nucleotide sequence of the calpain linker region of pAP-296 isreferred to herein as SEQ ID NO. 124.

The DNA sequence of the pAP-296 insert is referred to herein as SEQ IDNO. 125.

The amino acid sequence of the mutant preproricin linker region forcalpain, pAP-296, is referred to herein as SEQ ID NO. 126.

The amino acid sequence of the wild type linker region is referred toherein as SEQ ID NO. 127.

The nucleic acid molecule of the invention has sequences encoding an Achain of a ricin-like toxin, a B chain of a ricin-like toxin and aheterologous linker sequence containing a cleavage recognition site fora disease-specific protease. The nucleic acid may be expressed toprovide a recombinant protein having an A chain of a ricin-like toxin, aB chain of a ricin-like toxin and a heterologous linker sequencecontaining a cleavage recognition site for a disease-specific protease.

The nucleic acid molecule may comprise the A and/or B chain of ricin.The ricin gene has been cloned and sequenced, and the X-ray crystalstructures of the A and B chains are published (Rutenber, E., et al.Proteins 10:240-250 (1991); Weston et al., Mol. Biol. 244:410-422(1994); Lamb and Lord, Eur. J. Biochem. 14:265 (1985); Halling, K., etal., Nucleic Acids Res. 13:8019 (1985)). It will be appreciated that theinvention includes nucleic acid molecules encoding truncations of A andB chains of ricin like proteins and analogs and homologs of A and Bchains of ricin-like proteins and truncations thereof (i.e., ricin-likeproteins), as described herein. It will further be appreciated thatvariant forms of the nucleic acid molecules of the invention which ariseby alternative splicing of an mRNA corresponding to a cDNA of theinvention are encompassed by the invention.

Another aspect of the invention provides a nucleotide sequence whichhybridizes under high stringency conditions to a nucleotide sequenceencoding the A and/or B chains of a ricin-like protein. Appropriatestringency conditions which promote DNA hybridization are known to thoseskilled in the art, or can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example,6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by awash of 2.0×SSC at 50° C. may be employed. The stringency may beselected based on the conditions used in the wash step. By way ofexample, the salt concentration in the wash step can be selected from ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be at high stringency conditions, at about 65° C.

The nucleic acid molecule may comprise the A and/or B chain of aricin-like toxin. Methods for cloning ricin-like toxins are known in theart and are described, for example, in E.P. 466,222. Sequences encodingricin or ricin-like A and B chains may be obtained by selectiveamplification of a coding region, using sets of degenerative primers orprobes for selectively amplifying the coding region in a genomic or cDNAlibrary. Appropriate primers may be selected from the nucleic acidsequence of A and B chains of ricin or ricin-like toxins. It is alsopossible to design synthetic oligonucleotide primers from the nucleotidesequences for use in PCR. Suitable primers may be selected from thesequences encoding regions of ricin-like proteins which are highlyconserved, as described for example in U.S. Pat. No. 5,101,025 and E.P.466,222.

A nucleic acid can be amplified from cDNA or genomic DNA using theseoligonucleotide primers and standard PCR amplification techniques. Thenucleic acid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. It will be appreciated that cDNAmay be prepared from mRNA, by isolating total cellular mRNA by a varietyof techniques, for example, by using the guanidinium-thiocyanateextraction procedure of Chirgwin et al., Biochemistry 18, 5294-5299(1979). cDNA is then synthesized from the mRNA using reversetranscriptase (for example, Moloney MLV reverse transcriptase availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase availablefrom Seikagaku America, Inc., St. Petersburg, Fla.). It will beappreciated that the methods described above may be used to obtain thecoding sequence from plants, bacteria or fungi, preferably plants, whichproduce known ricin-like proteins and also to screen for the presence ofgenes encoding as yet unknown ricin-like proteins.

A sequence containing a cleavage recognition site for a specificprotease may be selected based on the disease or the pathogen which isto be targeted by the recombinant protein. The cleavage recognition sitemay be selected from sequences known to encode a cleavage recognitionsite for the cancer, viral or parasitic protease. Sequences encodingcleavage recognition sites may be identified by testing the expressionproduct of the sequence for susceptibility to cleavage by the respectiveprotease.

A sequence containing a cleavage recognition site for a viral, fungal,parasitic or cancer associated protease may be selected based on theretrovirus which is to be targeted by the recombinant protein. Thecleavage recognition site may be selected from sequences known to encodea cleavage recognition site for the viral, fungal, parasitic or cancerassociated protease. Sequences encoding cleavage recognition sites maybe identified by testing the expression product of the sequence forsusceptibility to cleavage by a viral, fungal, parasitic or cancerassociated protease. A polypeptide containing the suspected cleavagerecognition site may be incubated with a protease and the amount ofcleavage product determined (DiIannit, 1990, J. Biol. Chem. 285:17345-17354 (1990)).

The protease may be prepared by methods known in the art and used totest suspected cleavage recognition sites.

In one embodiment, the preparation of tumour-associated cathepsin B, itssubstrates and enzymatic activity assay methodology have been describedby Sloane, B. F. et al. (Proc. Natl. Acad. Sci. USA 83:2483-2487(1986)), Schwartz, M. K. (Clin. Chim. Acta 237:67-78 (1995)), andPanchal, R. G. et al. (Nature Biotechnol. 14:852-856 (1996)). Thepreparation of Epstein-Barr virus protease, its substrates and enzymaticactivity assay methodology have been described by Welch, A. R. (Proc.Natl. Acad. Sci. USA 88:10792-10796 (1991)).

In another embodiment, the preparation of Plasmondium falciparumproteases, their substrates and enzymatic activity assay methodologyhave been described by Goldberg, D. E. et al. (J. Exp. Med. 173:961-969(1991)), Cooper & Bujard (Mol. Biochem. Parasitol. 56:151-160 (1992)),Nwagwu, M. et al. (Exp. Parasitol. 75:399-414 (1992)), Rosenthal, P. J.et al. (J. Clin. Invest. 91:1052-1056 (1993)), Blackman, M. J. et al.(Mol. Biochem. Parasitol. 62:103-114 (1995)).

In a further embodiment, the preparation of proteases from humancytomegalovirus, human herpes virus, varicalla zoster virus andinfectious laryngotracheitis virus have been taught by Liu F. & Roizman,B. (J. Virol. 65:5149-5156 (1991)) and Welch, A. R. (Proc. Natl. Acad.Sci. USA 88:10792-10796 (1991)). In addition, their respectivesubstrates and enzymatic activity assay methodologies are alsodescribed.

In another embodiment, the preparation of hepatitis A virus protease,its substrates and enzymatic activity assay methodology have beendescribed by Jewell, D. A. et al. (Biochemistry 31:7862-7869 (1992)).The preparation of poliovirus protease, its substrates and enzymaticactivity assay methodology have been described by Weidner, J. R. et al.(Arch. Biochem. Biophys. 286:402-408 (1991)). The preparation of humanrhinovirus protease, its substrates and enzymatic activity assaymethodology have been described by Long, A. C. et al. (FEBS Lett.258:75-78 (1989)).

In another embodiment of the invention, the preparation of proteasesassociated with Candida yeasts their substrates and enzymatic activityare contemplated, including the aspartic proteinases which have beenassociated specifically with numerous virulent strains of Candidaincluding Candida albican, Candida tropicalis, and Candida parapsilosis(Abad-Zapatero, C. et al., Protein Sci. 5:640-652 (1996); Cutfield, S.M. et al., Biochemistry 35:398-410 (1995); Ruchel, R. et al, Zentralbl.Bakteriol. Mikrobiol Hyg. I Abt. Orig. A. 255:537-548 (1983); Remold, H.et al., Biochim. Biophys. Acta 167:399-406 (1968)).

The nucleic acid molecule of the invention may be prepared by sitedirected mutagenesis. For example, the cleavage site of adisease-specific protease may be prepared by site directed mutagenesisof the homologous linker sequence of a proricin-like toxin. Proceduresfor cloning proricin-like genes, encoding a linker sequence aredescribed in EP 466,222. Site directed mutagenesis may be accomplishedby DNA amplification of mutagenic primers in combination with flankingprimers. Suitable procedures using the mutagenic primers are shown inParts A and B of FIGS. 1-4, FIGS. 13-16, FIGS. 18-36, FIGS. 38-41, andFIGS. 50-67.

The nucleic acid molecule of the invention may also encode a fusionprotein. A sequence encoding a heterologous linker sequence containing acleavage recognition site for a disease-specific protease may be clonedfrom a cDNA or genomic library or chemically synthesized based on theknown sequence of such cleavage sites. The heterologous linker sequencemay then be fused in frame with the sequences encoding the A and Bchains of the ricin-like toxin for expression as a fusion protein. Itwill be appreciated that a nucleic acid molecule encoding a fusionprotein may contain a sequence encoding an A chain and a B chain fromthe same ricin-like toxin or the encoded A and B chains may be fromdifferent toxins. For example, the A chain may be derived from ricin andthe B chain may be derived from abrin. A protein may also be prepared bychemical conjugation of the A and B chains and linker sequence usingconventional coupling agents for covalent attachment.

An isolated and purified nucleic acid molecule of the invention which isRNA can be isolated by cloning a cDNA encoding an A and B chain and alinker into an appropriate vector which allows for transcription of thecDNA to produce an RNA molecule which encodes a protein of theinvention. For example, a cDNA can be cloned downstream of abacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can betranscribed in vitro with T7 polymerase, and the resultant RNA can beisolated by standard techniques.

Recombinant Protein of the Invention

As previously mentioned, the invention provides novel recombinantproteins which incorporate the A and B chains of a ricin like toxinlinked by a heterologous linker sequence containing a cleavagerecognition site for a disease-specific protease. It is an advantage ofthe recombinant proteins of the invention that they are non-toxic untilthe A chain is liberated from the B chain by specific cleavage of thelinker by the target protease.

Thus the protein may be used to specifically target cancer cells orcells infected with a virus or parasite in the absence of additionalspecific cell-binding components to target infected cells. It is afurther advantage that the disease-specific protease cleaves theheterologous linker intracellularly thereby releasing the toxic A chaindirectly into the cytoplasm of the cancer cell or infected cell. As aresult, said cells are specifically targeted and non-infected normalcells are not directly exposed to the activated free A chain.

Ricin is a plant derived ribosome inhibiting protein which blocksprotein synthesis in eukaryotic cells. Ricin may be derived from theseeds of Ricinus communis (castor oil plant). The ricin toxin is aglycosylated heterodimer with A and B chain molecular masses of 30,625Da and 31,431 Da respectively. The A chain of ricin has an N-glycosidaseactivity and catalyzes the excision of a specific adenine residue fromthe 28S rRNA of eukaryotic ribosomes (Endo, Y; & Tsurugi, K. J. Biol.Chem. 262:8128 (1987)). The B chain of ricin, although not toxic initself, promotes the toxicity of the A chain by binding to galactoseresidues on the surface of eukaryotic cells and stimulatingreceptor-mediated endocytosis of the toxin molecule (Simmons et al.,Biol. Chem. 261:7912 (1986)).

All protein toxins are initially produced in an inactive, precursorform. Ricin is initially produced as a single polypeptide (preproricin)with a 35 amino acid N-terminal presequence and 12 amino acid linkerbetween the A and B chains. The pre-sequence is removed duringtranslocation of the ricin precursor into the endoplasmic reticulum(Lord, J. M., Eur. J. Biochem. 146:403-409 (1985) and Lord, J. M., Eur.J. Biochem. 146:411-416 (1985)). The proricin is then translocated intospecialized organelles called protein bodies where a plant proteasecleaves the protein at a linker region between the A and B chains (Lord,J. M. et al., FASAB Journal 8:201-208 (1994)). The two chains, however,remain covalently attached by an interchain disulfide bond (cysteine 259in the A chain to cysteine 4 in the B chain) and mature disulfide linkedricin is stored in protein bodies inside plant cells. The A chain isinactive in the proricin (O'Hare, M., et al., FEBS Lett. 273:200-204(1990)) and it is inactive in the disulfide-linked mature ricin(Richardson, P. T. et al., FEBS Lett. 255:15-20 (1989)). The ribosomesof the castor bean plant are themselves susceptible to inactivation byricin A chain; however, as there is no cell surface galactose to permitB chain recognition the A chain cannot re-enter the cell.

Ricin-like proteins include, but are not limited to, bacterial, fungaland plant toxins which have A and B chains and inactivate ribosomes andinhibit protein synthesis. The A chain is an active polypeptide subunitwhich is responsible for the pharmacologic effect of the toxin. In mostcases the active component of the A chain is an enzyme. The B chain isresponsible for binding the toxin to the cell surface and is thought tofacilitate entry of the A chain into the cell cytoplasm. The A and Bchains in the mature toxins are linked by disulfide bonds. The toxinsmost similar in structure to ricin are plant toxins which have one Achain and one B chain. Examples of such toxins include abrin which maybe isolated from the seeds of Abrus precatorius and modeccin, volkensinand viscumin.

Ricin-like bacterial proteins include diphtheria toxin, which isproduced by Corynebacterium diphtheriae, Pseudomonas enterotoxin A andcholera toxin. It will be appreciated that the term ricin-like toxins isalso intended to include the A chain of those toxins which have only anA chain. The recombinant proteins of the invention could include the Achain of these toxins conjugated to, or expressed as, a recombinantprotein with the B chain of another toxin. Examples of plant toxinshaving only an A chain include trichosanthin, MMC and pokeweed antiviralproteins, dianthin 30, dianthin 32, crotin II, curcin II and wheat germinhibitor. Examples of fungal toxins having only an A chain includealpha-sarcin, restrictocin, mitogillin, enomycin, phenomycin. Examplesof bacterial toxins having only an A chain include cytotoxin fromShigella dysenteriae and related Shiga-like toxins. Recombinanttrichosanthin and the coding sequence thereof is disclosed in U.S. Pat.Nos. 5,101,025 and 5,128,460.

In addition to the entire A or B chains of a ricin-like toxin, it willbe appreciated that the recombinant protein of the invention may containonly that portion of the A chain which is necessary for exerting itscytotoxic effect. For example, the first 30 amino acids of the ricin Achain may be removed resulting in a truncated A chain which retainstoxic activity. The truncated ricin or ricin-like A chain may beprepared by expression of a truncated gene or by proteolyticdegradation, for example with Nagarase (Funmatsu et al., Jap. J. Med.Sci. Biol. 23:264-267 (1970)). Similarly, the recombinant protein of theinvention may contain only that portion of the B chain necessary forgalactose recognition, cell binding and transport into the cellcytoplasm. Truncated B chains are described for example in E.P. 145,111.The A and B chains may be glycosylated or non-glycosylated. GlycosylatedA and B chains may be obtained by expression in the appropriate hostcell capable of glycosylation. Non-glycosylated chains may be obtainedby expression in nonglycosylating host cells or by treatment to removeor destroy the carbohydrate moieties.

The proteins of the invention may be prepared using recombinant DNAmethods. Accordingly, the nucleic acid molecules of the presentinvention may be incorporated in a known manner into an appropriateexpression vector which ensures good expression of the protein. Possibleexpression vectors include but are not limited to cosmids, plasmids, ormodified viruses (e.g. replication defective retroviruses, adenovirusesand adeno-associated viruses), so long as the vector is compatible withthe host cell used. The expression vectors are “suitable fortransformation of a host cell”, which means that the expression vectorscontain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector. It will also be appreciated that thenecessary regulatory sequences may be supplied by the native A and Bchains and/or its flanking regions.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformant host cell. The term “transformant host cell” isintended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells. Other suitable host cells can be foundin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1991).

More particularly, bacterial host cells suitable for carrying out thepresent invention include E. coli, B. subtilis, Salmonella typhimurium,and various species within the genus' Pseudomonas, Streptomyces, andStaphylococcus, as well as many other bacterial species well known toone of ordinary skill in the art. Suitable bacterial expression vectorspreferably comprise a promoter which functions in the host cell, one ormore selectable phenotypic markers, and a bacterial origin ofreplication. Representative promoters include the β-lactamase(penicillinase) and lactose promoter system (see Chang et al., Nature275:615 (1978)), the trp promoter (Nichols and Yanofsky, Meth inEnzymology 101:155, (1983) and the tac promoter (Russell et al., Gene20: 231, (1982)). Representative selectable markers include variousantibiotic resistance markers such as the kanamycin or ampicillinresistance genes. Suitable expression vectors include but are notlimited to bacteriophages such as lambda derivatives or plasmids such aspBR322 (Bolivar et al., Gene 2:9S, (1977)), the pUC plasmids pUC18,pUC19, pUC118, pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983and Vieira and Messing, Gene 19:259-268 (1982)), and pNH8A, pNH16a,pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.). Typicalfusion expression vectors which may be used are discussed above, e.g.pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.). Examples ofinducible non-fusion expression vectors include pTrc (Amann et al., Gene69:301-315 (1988)) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego,Calif., 60-89 (1990)).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerivisaeinclude pYepSecl (Baldari. et al., Embo J. 6:229-234 (1987)), pMFa(Kurjan and Herskowitz, Cell 30:933-943 (1982)), pJRY88 (Schultz et al.,Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Protocols for the transformation of yeast and fungi are wellknown to those of ordinary skill in the art.(see Hinnen et al., Proc.Nati. Acad. Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology153:163 (1983), and Cullen et al. (Bio/Technology 5:369 (1987)).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573) and NS-1 cells. Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58(1987), which reviews the use of Agrobacterium rhizogenes vectors; seealso Zambryski et al., Genetic Engineering, Principles and Methods,Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, NewYork (1984), which describes the use of expression vectors for plantcells, including, among others, pAS2022, pAS2023, and pAS2034).

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., Mol.Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow, V. A., andSummers, M. D., Virology 170:31-39 (1989)). Some baculovirus-insect cellexpression systems suitable for expression of the recombinant proteinsof the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as, rats, rabbits, sheep and pigs(Hammer et al. Nature 315:680-683 (1985); Palmiter et al. Science222:809-814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA82:4438-4442 (1985); Palmiter and Brinster Cell 41:343-345 (1985) andU.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964)) orsynthesis in homogenous solution (Houbenweyl, Methods of OrganicChemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)).

The present invention also provides proteins comprising an A chain of aricin-like toxin, a B chain of a ricin-like toxin and a heterologouslinker amino acid sequence linking the A and B chains, wherein thelinker sequence contains a cleavage recognition site for adisease-specific protease. Such a protein could be prepared other thanby recombinant means, for example by chemical synthesis or byconjugation of A and B chains and a linker sequence isolated andpurified from their natural plant, fungal or bacterial source. Such Aand B chains could be prepared having the glycosylation pattern of thenative ricin-like toxin.

N-terminal or C-terminal fusion proteins comprising the protein of theinvention conjugated with other molecules, such as proteins may beprepared by fusing, through recombinant techniques. The resultant fusionproteins contain a protein of the invention fused to the selectedprotein or marker protein as described herein. The recombinant proteinof the invention may also be conjugated to other proteins by knowntechniques. For example, the proteins may be coupled usingheterobifunctional thiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins which may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Utility of the Nucleic Acid Molecules and Proteins of the Invention

The proteins of the invention may be used to specifically inhibit ordestroy mammalian cells affected by a disease or infection which haveassociated with such cells a specific protease, i.e., disease-specific,for example cancer cells or cells infected with a virus, fungus orparasite, all of which are encompased within the term“disease-specific.” It is an advantage of the recombinant proteins ofthe invention that they have specificity for said cells without the needfor a cell binding component. The ricin-like B chain of the recombinantproteins recognize galactose moieties on the cell surface and ensurethat the protein is taken up by the diseased cell and released into thecytoplasm. When the protein is internalized into a non-infected cell,cleavage of the heterologous linker would not occur in the absence ofthe disease-specific protease and the A chain will remain inactive boundto the B chain. Conversely, when the protein is internalized into adiseased cell, the disease-specific protease will cleave the cleavagerecognition site in the linker thereby releasing the toxic A chain.

The specificity of a recombinant protein of the invention may be testedby treating the protein with the disease-specific protease which isthought to be specific for the cleavage recognition site of the linkerand assaying for cleavage products. Disease-specific proteases may beisolated from cancer cells or infected cells, or they may be preparedrecombinantly, for example following the procedures in Darket et al. (J.Biol. Chem. 254:2307-2312 (1988)). The cleavage products may beidentified for example based on size, antigenicity or activity. Thetoxicity of the recombinant protein may be investigated by subjectingthe cleavage products to an in vitro translation assay in cell lysates,for example using Brome Mosaic Virus mRNA as a template. Toxicity of thecleavage products may be determined using a ribosomal inactivation assay(Westby et al., Bioconjugate Chem. 3:377-382 (1992)). The effect of thecleavage products on protein synthesis may be measured in standardizedassays of in vitro translation utilizing partially defined cell freesystems composed for example of a reticulocyte lysate preparation as asource of ribosomes and various essential cofactors, such as mRNAtemplate and amino acids. Use of radiolabelled amino acids in themixture allows quantitation of incorporation of free amino acidprecursors into trichloroacetic acid precipitable proteins. Rabbitreticulocyte lysates may be conveniently used (O'Hare, FEBS Lett.273:200-204 (1990)).

The ability of the recombinant proteins of the invention to selectivelyinhibit or destroy animal cancer cells or cells infected with a virus orparasite may be readily tested in vitro using animal cancer cell linesor cell cultures infected with the virus or parasite of interest. Theselective inhibitory effect of the recombinant proteins of the inventionmay be determined, for example, by demonstrating the selectiveinhibition of viral antigen expression in infected mammalian cells, theselective inhibition of general mRNA translation and protein synthesisin diseased cells, or selective inhibition of cellular proliferation incancer cells or infected cells.

Toxicity may also be measured based on cell viability, for example theviability of infected and non-infected cell cultures exposed to therecombinant protein may be compared. Cell viability may be assessed byknown techniques, such as trypan blue exclusion assays.

In another example, a number of models may be used to test thecytotoxicity of recombinant proteins having a heterologous linkersequence containing a cleavage recognition site for a cancer-associatedmatrix metalloprotease. Thompson, E. W. et al. (Breast Cancer Res.Treatment 31:357-370 (1994)) has described a model for the determinationof invasiveness of human breast cancer cells in vitro by measuringtumour cell-mediated proteolysis of extracellular matrix and tumour cellinvasion of reconstituted basement membrane (collagen, laminin,fibronectin, Matrigel or gelatin). Other applicable cancer cell modelsinclude cultured ovarian adenocarcinoma cells (Young, T. N. et al.Gynecol. Oncol. 62:89-99 (1996); Moore, D. H. et al. Gynecol. Oncol.65:78-82 (1997)), human follicular thyroid cancer cells (Demeure, M. J .et al., World J. Surg. 16:770-776 (1992)), human melanoma (A-2058) andfibrosarcoma (HT-1080) cell lines (Mackay, A. R. et al. Lab. Invest.70:781-783 (1994)), and lung squamous (HS-24) and adenocarcinoma (SB-3)cell lines (Spiess, E. et al. J. Histochem. Cytochem. 42:917-929(1994)). An in vivo test system involving the implantation of tumoursand measurement of tumour growth and metastasis in athymic nude mice hasalso been described (Thompson, E. W. et al., Breast Cancer Res.Treatment 31:357-370 (1994); Shi, Y. E. et al., Cancer Res. 53:1409-1415(1993)).

A further model may be used to test the cytotoxicity of recombinantproteins having a heterologous linker sequence containing a cleavagerecognition site for a cancer-associated Cathepsin B protease isprovided in human glioma (Mikkelsen, T. et al. J. Neurosurge, 83:285-290(1995)).

Similarly, the cytotoxicity of recombinant proteins having aheterologous linker sequence containing a cleavage recognition site fora malarial protease may be tested by a Plasmodium invasion assay usinghuman erythrocytes infected with mature-stage merozoite parasites asdescribed by McPherson, R. A. et al. (Mol. Biochem. Parasitol.62:233-242 (1993)). Alternatively, in vitro cultures of human hepaticparenchymal cells may be used to evaluate schizont infectivity andPlasmodium merozoite generation.

With respect to models of viral infection and replication, suitableanimal cells which can be cultured in vitro and which are capable ofmaintaining viral replication can be used as hosts. The toxicity of therecombinant protein for infected and non-infected cultures may then becompared. The ability of the recombinant protein of the invention toinhibit the expression of these viral antigens may be an importantindicator of the ability of the protein to inhibit viral replication.Levels of these antigens may be measured in assays using labelledantibodies having specificity for the antigens. Inhibition of viralantigen expression has been correlated with inhibition of viralreplication (U.S. Pat. No. 4,869,903). Toxicity may also be assessedbased on a decrease in protein synthesis in target cells, which may bemeasured by known techniques, such as incorporation of labelled aminoacids, such as [3H] leucine (O'Hare et al., FEBS Lett. 273:200-204(1990)). Infected cells may also be pulsed with radiolabelled thymidineand incorporation of the radioactive label into cellular DNA may betaken as a measure of cellular proliferation. Toxicity may also bemeasured based on cell death or lysis, for example, the viability ofinfected and non-infected cell cultures exposed to the recombinantprotein may be compared. Cell viability may be assessed by knowntechniques, such as trypan blue exclusion assays.

Although the primary specificity of the proteins of the invention fordiseased cells is mediated by the specific cleavage of the cleavagerecognition site of the linker, it will be appreciated that specificcell binding components may optionally be conjugated to the proteins ofthe invention. Such cell binding components may be expressed as fusionproteins with the proteins of the invention or the cell bindingcomponent may be physically or chemically coupled to the proteincomponent. Examples of suitable cell binding components includeantibodies to cancer, viral or parasitic proteins.

Antibodies having specificity for a cell surface protein may be preparedby conventional methods. A mammal, (e.g. a mouse, hamster, or rabbit)can be immunized with an immunogenic form of the peptide which elicitsan antibody response in the mammal. Techniques for conferringimmunogenicity on a peptide include conjugation to carriers or othertechniques well known in the art. For example, the peptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassay procedures can be used with theimmunogen as antigen to assess the levels of antibodies. Followingimmunization, antisera can be obtained and, if desired, polyclonalantibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art, (e.g. the hybridoma technique originally developed by Kohlerand Milstein (Nature 256:495-497 (1975)) as well as other techniquessuch as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4:72 (1983)), the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies in CancerTherapy Allen R., Bliss, Inc., pages 77-96 (1985)), and screening ofcombinatorial antibody libraries (Huse et al., Science 246:1275 (1989)).Hybridoma cells can be screened immunochemically for production ofantibodies specifically reactive with the peptide and the monoclonalantibodies can be isolated.

The term “antibody” as used herein is intended to include fragmentsthereof which also specifically react with a cell surface component.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above.For example, F(ab′)2 fragments can be generated by treating antibodywith pepsin. The resulting F(ab′)2 fragment can be treated to reducedisulfide bridges to produce Fab′ fragments.

Chimeric antibody derivatives, i.e., antibody molecules that combine anon-human animal variable region and a human constant region are alsocontemplated within the scope of the invention. Chimeric antibodymolecules can include, for example, the antigen binding domain from anantibody of a mouse, rat, or other species, with human constant regions.Conventional methods may be used to make chimeric antibodies containingthe immunoglobulin variable region which recognizes a cell surfaceantigen (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A.81:6851 (1985); Takeda et al., Nature 314:452 (1985), Cabilly et al.,U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchiet al., E.P. Patent No. 171,496; European Patent No. 173,494, UnitedKingdom Patent No. GB 2177096B). It is expected that chimeric antibodieswould be less immunogenic in a human subject than the correspondingnon-chimeric antibody.

Monoclonal or chimeric antibodies specifically reactive against cellsurface components can be further humanized by producing human constantregion chimeras, in which parts of the variable regions, particularlythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such immunoglobulin molecules may be made by techniques known in theart, (e.g. Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312(1983); Kozbor et al., Immunology Today 4:7279 (1983); Olsson et al.,Meth, Enzymol., 92:3-16 (1982), and PCT Publication W092/06193 or EP239,400). Humanized antibodies can also be commercially produced(Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)

Specific antibodies, or antibody fragments, reactive against cellsurface components may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with cell surface components. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries (See for example Ward et al., Nature341:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); andMcCafferty et al., Nature 348:552-554 (1990)). Alternatively, a SCID-humouse, for example the model developed by Genpharm, can be used toproduce antibodies, or fragments thereof.

The proteins of the invention may be formulated into pharmaceuticalcompositions for adminstration to subjects in a biologically compatibleform suitable for administration in vivo. By “biologically compatibleform suitable for administration in vivo” is meant a form of thesubstance to be administered in which any toxic effects are outweighedby the therapeutic effects. The substances may be administered to livingorganisms including humans, and animals. Administration of atherapeutically active amount of the pharmaceutical compositions of thepresent invention is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result. For example, atherapeutically active amount of a substance may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of antibody to elicit a desired response inthe individual. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, everal divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The nucleic acid molecules of the invention may be formulated intopharmaceutical compositions for adminstration to subjects in abiologically compatible form suitable for administration in vivo. By“biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects. The substances may beadministered to living organisms including humans, and animals.Administration of a therapeutically active amount of the pharmaceuticalcompositions of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. For example, a therapeutically active amount of a substance mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of antibody to elicit adesired response in the individual. Dosage regime may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

The active substance may be administered in a convenient manner such asby injection (subcutaneous, intravenous, intramuscular, etc.), oraladministration, inhalation, transdermal administration (such as topicalcream or ointment, etc.), or suppository applications. Depending on theroute of administration, the active substance may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions which may inactivate the compound.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe substances in association with one or more pharmaceuticallyacceptable vehicles or diluents, and contained in buffered solutionswith a suitable pH and iso-osmotic with the physiological fluids.

The pharmaceutical compositions may be used in methods for treatinganimals, including mammals, preferably humans, with cancer or infectedwith a virus or a parasite. It is anticipated that the compositions willbe particularly useful for treating patients with B-celllymphoproliferative disease, (melanoma), mononucleosis, cytomegalicinclusion disease, malaria, herpes, shingles, hepatitis, poliomyelitis,or infectious laryngotracheitis. The dosage and type of recombinantprotein to be administered will depend on a variety of factors which maybe readily monitored in human subjects. Such factors include theetiology and severity (grade and stage) of neoplasia, the stage ofmalarial infection (e.g. exoerythrocytic vs. erythrocytic), or antigenlevels associated with viral load in patient tissues or circulation.

As mentioned above, the novel recombinant toxic proteins and nucleicacid molecules of the present invention are useful in treating cancerousor infected cells wherein the cells contain a specific protease that cancleave the linker region of the recombinant toxic protein. One skilledin the art can appreciate that many different recombinant toxic proteinscan be prepared once a disease associated protease has been identified.For example,the novel recombinant toxic proteins and nucleic acidmolecules of the invention may be used to treat CNS tumors. Muller etal. (1993) describe increased activity of Insulin-type Growth FactorBinding Protein-3 (IGFBP-3) protease in the Cerebral Spinal Fluid ofpatients with CNS tumors. Cohen et al. (1992) claim thatprostate-specific antigen (PSA) is an IGFBP-3 protease. The pAP290construct described above is a substrate for PSA. Conover et al. (1994)claim that cathepsin D is IGFBP-3 protease. The pAP276 described hereinis a substrate for cathepsin D. Another example of a specific use of theinvention is treatment of human glioma which has been shown to producecathepsin D (Mikkelsen, T. et al. J. Neurosurge, 83:285-290 (1995)). ThepAP 214 and 272 define herein are substrates for cathepsin B.

In addition, the novel proteins and nucleic acid molecules of thepresent invention may be used to treat cystic fibrosis. Hansen et al.(1995) describe how CF airway disease is characterized byneutrophil-dominated chronic inflammation with an excess of uninhibitedneutrophil elastase (NE). NE levels in CF sputum are 350 times higherthan that found in normal sputum. The pAP294 described herein is asubstrate for neutrophil elastase.

As well, the novel proteins and nucleic acid molecules of the presentinvention may also be used to treat multiple sclerosis. Bever Jr. et al.(1994) implicate cathepsin B (possibly from inflammatory cells ofhematogenous origin) in the demyelination found in multiple sclerosis.pAPs 214 and 272 defined herein present substrates for cathepsin B.

The term “animal” as used herein includes all members of the animalkingdom including mammals, preferably humans.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Cloning and Expression of Proricin Variants Activatedby Disease-Specific Proteases

Isolation of total RNA

The preproricin gene was cloned from new foliage of the castor beanplant. Total messenger RNA was isolated according to establishedprocedures (Sambrook et al., Molecular Cloning: A Lab Manual (ColdSpring Harbour Press, Cold Spring Harbour, (1989)) and cDNA generatedusing reverse transcriptase.

cDNA Synthesis:

Oligonucleotides, corresponding to the extreme 5′ and 3′ ends of thepreproricin gene were synthesized and used to PCR amplify the gene.Using the cDNA sequence for preproricin (Lamb et al., Eur. J. Biochem.,145:266-270, 1985), several oligonucleotide primers were designed toflank the start and stop codons of the preproricin open reading frame.The oligonucleotides were synthesized using an Applied Biosystems Model392 DNA/RNA Synthesizer. First strand cDNA synthesis was primed usingthe oligonucleotide Ricin1729C (Table 1). Three micrograms of total RNAwas used as a template for oligo Ricin1729C primed synthesis of cDNAusing Superscript II Reverse Transcriptase (BRL) following themanufacturer's protocol.

DNA Amplification and Cloning

The first strand cDNA synthesis reaction was used as template for DNAamplification by the polymerase chain reaction (PCR). The preproricincDNA was amplified using the upstream primer Ricin-99 and the downstreamprimer Ricin1729C with Vent DNA polymerase (New England Biolabs) usingstandard procedures (Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)).Amplification was carried out in a Biometra thermal cycler(TRIO-Thermalcycler) using the following cycling parameters:denaturation 95° C. for 1 min., annealing 52° C. for 1 min., andextension 72° C. for 2 min., (33 cycles), followed by a final extensioncycle at 72° C. for 10 min. The 1846bp amplified product wasfractionated on an agarose gel (Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press,1989), and the DNA purified from the gel slice using Qiaex resin(Qiagen) following the manufacturer's protocol. The urified PCR fragmentencoding the preproricin cDNA was then ligated (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor Laboratory Press, 1989)) into an Eco RV-digested pBluescript IISK plasmid (Stratagene), and used to transform competent XL1-Blue cells(Stratagene). Positive clones were confirmed by restriction digestion ofpurified plasmid DNA. Plasmid DNA was extracted using a Qiaprep SpinPlasmid Miniprep Kit (Qiagen).

DNA Sequencing

The cloned PCR product containing the putative preproricin gene wasconfirmed by DNA sequencing of the entire cDNA clone (pAP-144).Sequencing was performed using an Applied Biosystems 373A Automated DNASequencer, and confirmed by double-stranded dideoxy sequencing by theSanger method using the Sequenase kit (USB). The oligonucleotide primersused for sequencing were as follows: Ricin267, Ricin486, Ricin725,Ricin937, Ricin1151, Ricini1399, Ricin1627, T3 primer(5′AATTAACCCTCACTAAAGGG-3′) (SEQ ID NO. 128) and T7 primer(5′GTAATACGACTCACTATAGGGC-3) (SEQ ID NO. 129). Sequence data wascompiled and analyzed using PC Gene software package (intelligenetics).The sequences and location of oligonucleotide primers is shown inTable 1. The oligonucleotide primers shown in Table 1 have been assignedthe following sequence ID numbers:

Ricin-109 is referred to herein as SEQ ID NO. 130;

Ricin-99Eco is referred to herein as SEQ ID NO. 131;

Ricin267 is referred to herein as SEQ ID NO. 132;

Ricin486 is referred to herein as SEQ ID NO. 133;

Ricin725 is referred to herein as SEQ ID NO. 134;

Ricin 937 is referred to herein as SEQ ID NO. 135;

Ricin 1151 is referred to herein as SEQ ID NO. 136;

Ricin 1399 is referred to herein as SEQ ID NO. 137;

Ricin 1627 is referred to herein as SEQ ID NO. 138;

Ricin 1729C is referred to herein as SEQ ID NO. 139; and

Ricin 1729C Xba is referred to herein as SEQ ID NO. 140.

Production and Cloning of Linker Variants

pAP144 cut with EcoRi was used as target for PCR pairs employing theRicin109-Eco oligonucleotide (Ricin-109Eco primer:5-GGAGGAATCCGGAGATGAAACCGGGAGGAAATACTATTGTAAT-3 (SEQ ID No. 141)) and amutagenic primer for the 5′ half of the linker as well as theRicin1729PstI primer (Ricin1729-PstI:5-GTAGGCGCTGCAGATAACTTGCTGTCCTTTCAG-3 (SEQ ID No. 142)) and a mutagenicprimer for the 3′ half of the linker. The cycling conditions used forthe PCRs were 98 degrees C. for 2 min.; 98C 1 min., 52C 1 min., 72C 1min. 15 sec. (30 cycles); 72 degrees C. 10 min.; 4 degrees C. soak. ThePCR products were then digested by EcoRi and PstI respectively,electrophoresed on an agarose gel, and the bands purified by via glasswool spin columns. Triple ligations comprising the PCR product pairs(corresponding halves of the new linker) and pVL1393 vector digestedwith EcoRI and PstI were carried out. Recombinant clones were identifiedby restriction digests of plasmid miniprep DNA and the altered linkersconfirmed by DNA sequencing. See FIG. 45 as an example of the cloningstrategy. Recombinant clones were identified by restriction digests ofplasmid miniprep DNA and the altered linkers confirmed by DNAsequencing. Note that since all altered linker variants were cloneddirectly into the pVL1393 vector odd-numbered pAPs were no longerrequired or produced.

Isolation of Recombinant Baculoviruses

Insect cells S. frugiperda (Sf9), and Trichoplusia ni (Tn368 andBTI-TN-581-4 (High Five)) were maintained on EX-CELL 405 medium (JRHBiosciences) supplemented with 10% total calf serum (Summers et al., AManual of Methods of Baculovirus Vectors and Insect Cell CultureProcedures, (Texas Agricultural Experiment Station, 1987)). Twomicrograms of recombinant pVL1393 DNA was co-transfected with 0.5microgram of BaculoGold AcNPV DNA (Pharmingen) into 2×10⁶ Tn368 insectcells following the manufacturer's protocol (Gruenwald et al.,Baculovirus Expression Vector System: Procedures and Methods Manual, 2ndEdition, (San Diego, Calif., 1993)). On day 5 post-transfection, mediawere centrifuged and the supernatants tested in limiting dilution assayswith Tn368 cells (Summers et al., A Manual of Methods of BaculovirusVectors and Insect Cell Culture Procedures, (Texas AgriculturalExperiment Station, 5 1987)). Recombinant viruses in the supernatantswere then amplified by infecting Tn368 cells at a multiplicity ofinfection (moi) of 0.1, followed by collection of day 3 to 5supernatants. A total of three rounds of amplification were performedfor each recombinant following established procedures (Summers et al., AManual of Methods of Baculovirus Vectors and Insect Cell CultureProcedures, (Texas Agricultural Experiment Station, 1987 and Gruenwaldet al., Baculovirus Expression Vector System: Procedures and MethodsManual, 2nd Edition, (San Diego, Calif., 1993)).

Expression of Mutant Proricin

Recombinant baculoviruses were used to infect 1×10⁷ Tn368 or sf9 cellsat an moi of 9 in EX-CELL 405 media (JRH Biosciences) with 25 mMa-lactose in spinner flasks. Media supernatants containing mutantproricins were collected 3 or 4 days post-infection.

Example 2 Harvesting and Affinity Column Purification of Pro-ricinVariants

Protein samples were harvested three days post transfection. The cellswere removed by centrifuging the media at 8288 g for ten minutesusing aGS3 (Sorvall) centrifuge rotor. The supernatant was further clarified bycentrifuging at 25400 g using a SLA-1500 rotor (Sorvall) for 45 minutes.Protease inhibitor phenylmethylsulfonyl fluoride (Sigma) was slowlyadded to a final concentration of lmM. The samples were further preparedby adding lactose to a concentration of 20 mM (not including theprevious lactose contained in the expression medium). The samples wereconcentrated to 700 mL using a Prep/Scale-TPF Cartridge (2.5 ft, 10Kregenerated cellulose (Millipore)) and a Masterflex pump. The sampleswere then dialysed for 2 days in 1×Column Buffer (50 mM Tris, 100 mMNaCl, 0.02% NaN3, pH 7.5) using dialysis tubing (10 K MWCO, 32 mm flatwidth(Spectra/Por)). Subsequently, the samples were clarified by centrifuging at 25400 g using a SLA-1500 rotor (Sorvall) for 45 minutes.

Following centrifugation, the samples were degassed and applied at 4degrees C. to a XK26/20 (Pharmacia) column (attached to a Pharmaciaperistaltic pump, Pharmacia Single-path Monitor UV-1 Control and OpticalUnits, and Bromma LKB 2210 2-Channel Recorder) containing 20 mL ofa-Lactose Agarose Resin (Sigma). The column was washed for 3 hours with1×Column buffer. Elution of pro-ricin variant was performed by elutingwith buffer (1×Column buffer (0.1% NaN3), 100 mM Lactose) until thebaseline was again restored. The samples were concentrated using anAmicon 8050 concentrator (Amicon) with a YM10 76 mm membrane, utilizingargon gas to pressurize the chamber. The samples were furtherconcentrated in Centricon 10 (Millipore) concentrators according tomanufacturer's specifications.

Purification of Variant pAP-Protein by Gel Filtration Chromatography

In order to purify the pro-ricin variant from processed materialproduced during fermentation, the protein was applied to a SUPERDEX 75(16/60) column and SUPERDEX 200 (16/60) column (Pharmacia) connected inseries equilibrated with 50 mM Tris, 100 mM NaCl, pH 7.5 containing 100mM Lactose and 0.1% β-mercaptoethanol (βME). The flow rate of the columnwas 0.15 mL/min and fractions were collected every 25 minutes. The UV(280 nm) trace was used to determine the approximate location of thepurified pAP-protein and thus determine the samples for Westernanalysis.

Western Analysis of Column Fractions

Fractions eluted from the SUPERDEX columns (Pharmacia) were analyzed forpurity using standard Western blotting techniques. An aliquot of 10 μLfrom each fraction was boiled in 1×sample buffer (62.6 mM Tris-C1, pH6.8, 4.4% βME, 2% sodium dodecyl sulfate (SDS), 5% glycerol (all fromSigma) and 0.002% bromophenol blue (Biorad)) for five minutes. Denaturedsamples were loaded on 12% Tris-Glycine Gels (Biorad) along with 50 ngof RCA₆₀ (Sigma) and 5 μL of kaleidoscope prestained standards (Biorad).Electrophoresis was carried out for ninety minutes at 100V in 25 mMTris-Cl, pH 8.3, 0.1% SDS, and 192 mM glycine using the BioRad MiniProtean II cells (Biorad).

Following electrophoresis gels were equilibrated in transfer buffer (48mM Tris, 39 mM glycine, 0.0375% SDS, and 20% Methanol) for a fewminutes. PVDF Biorad membrane was presoaked for one minute in 100%methanol, rinsed in ddH₂O and two minutes in transfer buffer. Whatmanpaper was soaked briefly in transfer buffer. Five pieces of Whatmanpaper, membrane, gel, and another five pieces of Whatman paper werearranged on the bottom cathode (anode) of the Pharmacia Novablottransfer apparatus (Pharmacia). Transfer was for one hour at constantcurrent (2 mA/cm²).

Transfer was confirmed by checking for the appearance of the prestainedstandards on the membrane. Non-specific sites on the membrane wereblocked by incubating the blot for thirty minutes in 1×PhosphateBuffered Saline (1×PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.5 mMKH₂PO₄, pH 7.4) with 5% skim milk powder (Carnation). Primary antibody(Rabbit α-ricin, Sigma) was diluted 1:3000 in 1×PBS containing 0.1%Tween 20 (Sigma) and 2.5% skim milk and incubated with blot for fortyfive minutes on a orbital shaker (VWR). Non-specifically bound primaryantibody was removed by washing the blot for ten minutes with 1×PBScontaining 0.2% Tween 20. This was repeated four times. Secondaryantibody donkey anti-rabbit (Amersham) was incubated with the blot underthe same conditions as the primary antibody. Excess secondary antibodywas washed as described above. Blots were developed with the ECL WesternBlotting detection reagents according to the manufacturer'sinstructions. Blots were exposed to Medtec's Full Speed Blue Film(Medtee) or Amersham's ECL Hyperfilm (Amersham) for one second to fiveminutes. Film was developed in a KODAK Automatic Developer.

Determination of Lectin Binding Ability of Pro-ricin Variant

An Immulon 2 plate (VDVR) was coated with 100 μl per well of 10 g/ml ofasialofetuin and left overnight at 4° C. The plate was washed with 3×300μL per well with ddH₂O using an automated plate washer (BioRad). Theplate was blocked for one hour at 37° C. by adding 300 μL per well ofPBS containing 1% ovalbumin. The plate was washed again as above.Pro-ricin variant pAP-protein was added to the plate in variousdilutions in 1×Baculo. A standard curve of RCA₆₀ (Sigma) from 1-10 ngwas also included. The plate was incubated for 1 h at 37° C. The platewas washed as above. Anti-ricin monoclonal antibody (Sigma) was diluted1:3000 in 1×PBS containing 0.5% ovalbumin and 0.1% tween-20, added at100 μL per well and incubated for 1 h at 37° C. The plate was washed asabove. Donkey-anti rabbity polyclonal antibody was diluted 1:3000 in1×PBS containing 0.5% ovalbumin, 0.1% Tween-20, and added at 100 μL perwell and incubated for 1 h at 37° C. The plate was given a final wash asdescribed above. Substrate was added to plate at 100 μL per well (1mg/ml o-phenylenediamine (Sigma), 1 μL/ml H₂O₂, 25 μL of stop solution(20% H₂SO₄) was added and the absorbance read (A490 nm-A630 nm) using aSPECTRA MAX 340 plate reader (Molecular Devices).

Determination of pAP -Protein Activity Using the Rabbit ReticulocyteAssay

Ricin samples were prepared for reduction.

A) RCA₆₀=3,500 ng/μL of RCA₆₀+997 μL 1×Endo buffer (25 mM Tris, 25 mMKCl,5 mM MGCl₂, pH 7.6) Reduction=95 μL of 10 ng/μL+5 μLβ-mercaptoethanol

B) Ricin variants Reduction=40 μL variant+2 μL β-mercaptoethanol Thericin standard and the variants were incubated for 30 minutes at roomtemperature.

Ricin-Rabbit Reticulocyte Lysate Reaction

The required number of 0.5 mL tubes were labelled. (2 tubes for eachsample, + and − aniline). To each of the sample tubes 20 μL of 1×endobuffer was added, and 30 μL of buffer was added to the controls. To thesample tubes either 10 μL of 10 ng/μL Ricin or 10 μL of variant wasadded. Finally, 30 μL of rabbit reticulocyte lysate was added to all thetubes. The samples were incubated for 30 minutes at 30° C. using thethermal block. Samples were removed from the eppendorf tube and contentsadded into a 1.5 mL tube containing 1 mL of TRIZOL (Gibco). Samples wereincubated for 15 minutes at room temperature. After the incubation, 200μL of chloroform was added, and the sample was vortexed and spun at12,000 g for 15 minutes at 4° C. The top aqueous layer from the sampleswas removed and contents added to a 1 mL tube containing 500 μL ofisopropanol. Samples were incubated for 15 minutes at room temperatureand then centrifuged at 12,000 for 15 minutes at 4° C. Supernatant wasremoved and the pellets were washed with 1 mL of 70% ethanol.Centrifugation at 12,000 g for 5 minutes at 4° C. precipitated the RNA.All but approximately 20μL of the supernatant was removed and air dried.Pellets from the other samples (+aniline samples) were dissolved in 20μL of DEPC treated ddH₂O. An 80 μL aliquot of 1 M aniline (distilled)with 2.8 M acetic acid was added to these RNA samples and transferred toa fresh 0.5 mL tube. The samples were incubated in the dark for 3minutes at 60° C. RNA was precipitated by adding 100 μL of 95% ethanoland 5 μL of 3M sodium acetate, pH 5.2 to each tube and centrifuging at12,000 g for 30 minutes at 4° C. Pellets were washed with 1 mL 70%ethanol and centrifuged again at 12,000 g for 5 minutes at 4° C. toprecipitate RNA. The supernatant was removed and air dried. Thesepellets were dissolved in 10 μL of 0.1×E buffer. To all samples, 10 μLof formamide loading dye was added. The RNA ladder (8 μL of ladder+8 μLof loading dye) was also included. Samples were incubated for 2 minutesat 70° C. on the thermal block. Electrophoresis was carried out on thesamples using 1.2% agarose, 50% formamide gels in 0.1×E buffer+0.2% SDS.The gel was run for 90 minutes at 75 watts. RNA was visualized bystaining the gel in 1 μg/μL ethidium bromide in running buffer for 45minutes. The gel was examined on a 302 nm UV box, photographed using thegel documentation system and saved to a computer disk.

Results:

Protein Expression Yields

Aliquots were taken at each stop of the harvesting/purification andtested. Yields of functional ricin variant were determined by ELISA.Typical results of an 2400 mL prep of infected T. ni cells are givenbelow.

Aliquot μg pAP 220 Before concentration and dialysis 6000 Afterconcentration and dialysis 4931 alpha- Lactose agarose column flowthrough 219 alpha- Lactose agarose column elution 1058 Yield: 1058/6000= 17.6%

Purification of pAP-Protein and Western Analysis of Column Fractions

Partially purfied pAP-protein was applied to Superdex 75 and 200 (16/60)columns connected in series in order to remove the contaminatingnon-specifically processed pAP-protein. Eluted fractions were tested viaWestern analysis as described above and the fractions containing themost pure protein were pooled, concentrated and re-applied to thecolumn. The variant was applied a total of three times to the column.Final purified pAP-protein has less than 1% processed variant.

The purified pAP-protein was tested for susceptibility to cleavage bythe particular protease and for activation of the A-chain of thepro-ricin variant, (inhibition of protein synthesis). Typically,pAP-protein was incubated with and without protease for a specified timeperiod and then electrophoresed and blotted. Cleaved pAP will run as two30 kDa proteins (B is slightly larger) under reducing (SDS-PAGE)conditions. Unprocessed pAP-protein, which contains the linker region,will run at 60 kDa.

Activation of pAP-Protein Variant with Specific Protease

Activation of protease treated pAP-protein is based on the method of Mayet al. (EMBO Journal. 8 301-8, 1989). Activation of ricin A chain uponcleavage of the intermediary linker results in catalytic depurination ofthe adenosine 4325 residue of 28S or 26S rRNA. This depurination rendersthe molecule susceptible to amine-catalyzed hydrolysis by aniline of thephosphodiester bond on either side of the modification site. The resultis a diagnostic 390 base band. As such, reticulocyte ribosomes incubatedwith biochemically purified ricin A chain, released the characteristicRNA fragment upon aniline treatment of isolated rRNA (May, M. J. et al.Embo. Journal, 8:301-308 at 302-303 (1989)). It is on this basis thatthe assay allows for the determination of activity of a ricin A chainwhich has been cleaved from the intact unit containing a particularvariant linker sequence.

Example 3 In Vitro Protease Digestion of Proricin Variants

Affinity-purified proricin variant is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion. Ricin-like toxin variants are eluted from the lactose-agarosematrix in protease digestion buffer (50 mM NaCl, 50 mM Na-acetate, pH5.5, 1 mM dithiothreitol) containing 100 mM lactose. Proricin substrateis then incubated at 37° C. for 60 minutes with a disease-specificprotease. The cleavage products consisting ricin A and B chains areidentified using SDS/PAGE (Sambrook et al., Molecular Cloning: aLaboratory Manual, 2nd. ed., Cold Spring Harbor Press, 1989), followedby Western blot analysis using anti-ricin antibodies (Sigma).

Cathepsin B may be obtained from Medcor or Calbiochem. Matrixmetalloproteinases may be prepared substantially as described by Lark,M. W. et al. (Proceedings of the 4th International Conference of theImflammation Research Association Abstract 145 (1988)) and Welch, A. R.et al. (Arch. Biochem. Biophys. 324:59-64 (1995)). Candida acid proteasemay be prepared substantially as described in Remold, H. H. et al.(Biochim. Biophys. Acta 167:399406 (1968)), Ray, T. L. and Payne, C. D.(Infect. Immunol. 58:508-514 (1990)) and Fusek, M. et al. (FEBS Lett.327:108-112 (1993)). Hepatitis A protease may be prepared as describedin Jewell, D. A. et al. (Biochemistry 31:7862-7869 (1992)). Plasmodiumproteases may be prepared as described in Goldberg, D. E. et al. (J.Exp. Med. 173:961-969 (1991)) and Cooper, J. A. and Bujard, H. (Mol.Biochem. Parasitol. 56:151-160 (1992)).

In Vitro Cytotoxicity Assay:

Human ovarian cancer cells (e.g. MA148) are seeded in 96-wellflat-bottom plates and are exposed to ricin-like toxin variants orcontrol medium at 37° C. for 16 h. The viability of the cancer cells isdetermined by measuring [³⁵S]methionine incorporation and issignificantly lower in wells treated with the toxin variants than thosewith control medium.

In Vivo Tumour Growth Inhibition Assay:

Human breast cancer (e.g. MCF-7) cells are maintained in suitable mediumcontaining 10% fetal calf serum. The cells are grown, harvested andsubsequently injected subcutaneously into ovariectomized athymic nudemice. Tumour size is determined at intervals by measuring tworight-angle measurements using calipers. In animals that receivedricin-like toxin variants containing the matrixmetalloproteinase-sensitive linkers, tumour size and the rate of tumourgrowth are lower than animals in the control group.

In Vivo Tumour Metastasis Assay:

The metastasis study is performed substantially as described in Honn, K.V. et al. (Biochem. Pharmacol. 34:235-241 (1985)). Viable B16a melanomatumour cells are prepared and injected subcutaneously into the leftaxillary region of syngeneic mice. The extent of tumour metastasis ismeasured after 4 weeks. The lungs are removed from the animals and arefixed in Bouin's solution and macroscopic pulmonary metastases arecounted using a dissecting microscope. In general without therapeuticintervention, injection of 10⁵ viable tumour cells forms approximately40-50 pulmonary metastases. The number of metastases in animal treatedwith proricin variants containing cathepsin B-sensitive linkers issubstantially lower.

Example 4 In Vitro Protease Digestion of Proricin Variants by CancerProteases Cathepsin B or MMP-9

The general protocol for proricin digestion by cancer proteases isdescribed in Examples 2 and 3.

In Vitro Protease Digestion of Cathepsin B Proricin Variant

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion. The proricin substrate is digested in a Cathepsin B proteasebuffer (50 mM Sodium acetate, 2 mM EDTA, 0.05% Triton) at 40° C. Twohours and overnight (16 hr) digestion reactions are carried out using100 ng of proricin substrate and 100 and 618 ng of Cathepsin B proteaseper reaction (CALBIOCHEM, USA). The cleavage products of proricin (ricinA and B chains) are identified using SDS/PAGE (Sambrook et al.,Molecular cloning: a laboratory Manual, 2nd. ed., Cold Spring HarborPress, 1989), followed by Western blot analysis using anti-ricinantibodies (Sigma).

In Vitro Protease Digestion of MMP-9 Proricin Variant

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion. The proricin substrate is digested in 1×column buffer (100 mMNaCl, 50 mM Tris, PH 7.5) at 37° C. Two hours and overnight (16 hr)digestion reactions are set up using 50 ng of MMP-9 proricin substrateand 20 and 200 ng of MMP-9 protease per reaction (CALBIOCHEM, USA). Thecleavage products of proricin (ricin A and B chains) are identifiedusing SDS/PAGE (Sambrook et al., Molecular cloning: a laboratory Manual,2nd. ed., Cold Spring Harbor Press, 1989), followed by Western blotanalysis using anti-ricin antibodies (Sigma).

The protocol for Western analysis of ricin chains is described inExample 2.

Results

FIGS. 48 and 49 illustrate Western blots showing the cleavage of theprotease-sensitive linkers by cathepsin B (pAP 214) and MMP-9 (pAP 220)respectively. Without protease digestion, the proricin variant appearsas a single band at approximately 60 kDa (Lane B of FIG. 48 and Lane Aof FIG. 49). Wild type ricin A chain and B chain appear as two disparatebands at approximately 30 kDa (Lane A of FIG. 48 and Lane E of FIG. 49).Increasing extent of proricin cleavage can clearly be observed withincreasing protease concentration (Lanes C and D of FIG. 48 and LanesB-C of FIG. 49).

Example 5

In Vitro Protease Digestion of Various Proricin Variants by TheirCorresponding Proteases.

The general protocol for proricin digestion by coresponding proteaseswas as desribed in Examples 2 and 3 and should be considered inconnection with the digestions described below.

Cleavage of pAP-222 Protein with the Matrix Metalloproteinase 2 (MMP-2)

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion.

The pAP-222 protein sample (1.0 ug) was digested with the MMP-2 protease(1.0 ug) overnight at 37° C. The total volume of the digestion reactionwas 21.5 ul, and 0.250 ug of the reaction sample was loaded on a proteingel. The MMP-2 protease was purchased from Calbiochem-NovabiochemCorporation, USA.

Cleavage of pAP-248 Protein with the Human Cytomegalovirus (HCMV)Protease

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion.

The pAP-248 protein sample (1.19 ug) was digested with the HCMV protease(1.13 ug) overnight at 37° C. The total volume of the digestion was 10.5ul, and 0.279 ug of the reaction sample was loaded on a protein gel. TheHCMV was purchased from BACHEM Bioscience Inc., USA.

Cleavage of pAP-256 Protein with the Hepatitis A Virus 3C (HAV 3C)Protease

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion.

The pAP-256 protein sample (1.26 ug) was digested with the HAV 3Cprotease (5 ug) overnight at 37° C. The total volume of the digestionwas 12.5 ul, and 0.302 ug of the digestion sample was loaded on aprotein gel. The HAV 3C protease was a gift from Dr. G. Lawson fromBates Collage, Main, USA.

Cleavage of pAP-270 Protein with the Matrix Metalloproteinase 2 (MMP-2)

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion.

The pAP-270 protein sample (0.120 ug) was digested with the MMP-2protease (0.25 ug) overnight at 37° C. The total volume of the digestionreaction was 22.5 ul, and 0.106 ug of the reaction sample was loaded ona protein gel. The MMP-2 protease was purchased fromCalbiochem-Novabiochem Corporation, USA.

Cleavage of pAP-288 Protein with tPA Plasminogen Tissue Activator

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion. The pAP-288 protein sample (1.65 ug) was digested with the t-PAprotease (0.5 ug) overnight at 37° C. The total volume of the digestionreaction was 55 ul, and 0.6 ug of the reaction sample was loaded on aprotein gel. The t-PA was purchased from Sigma Chemical Co., USA.

Cleavage of pAP-294 Protein with Human Neutraphil Elastase

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion.

The pAP-256 protein sample (0.6 ug) was digested with the Elastaseprotease (5 ug) at 25° C. for one hour. The total volume of thedigestion reaction was 52.5 ul, and 0.171 ug of the digestion sample wasloaded on a protein gel. The Human Neutrophil Elastase protease waspurchased from Cedarlane Laboratories Limited, Canada.

Cleavage of pAP-296 Protein with Calpain

Affinity-purified mutant proricin is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion. The pAP-296 protein sample (2.05 ug) was digested with theCalpain protease (10 ug) overnight at 37° C. The total volume of thedigestion reaction was 35 ul and 0.761 ug of the reaction sample wasloaded on a protein gel. The Calpain protease was purchased from SigmaChemical Co., USA

Results

FIGS. 52, 54, 58 & 66(MMP-2), 60, 64 and 62 show the cleavage ofproteases of linkers by HCMV, HAV 3C, MMP-2, t-PA, calpain, and humanneutraphil elastase respectively. Without protease digestion, theproricin variants appear as a single band at approximately 60 kDA (LaneA in connection with FIG. 52; Lane B of FIG. 54; Lane A of FIG. 58; LaneB of FIG. 60; and Lane C of FIG. 62; lane B of FIG. 64 and lane B ofFIG. 66). Wild type ricin chain A and B appear as two bands atapproximately 30 kDA (see for example Lanes C and D of FIG. 52) proricincleavage can clearly be obvserved with the appearance of 30 kDA bands inconnection with the protein which has been digested by the respectiveprotease (see Lane B of FIG. 52; Lane C of FIG. 54; or Lane B of FIG. 58for examples).

Example 6 In Vitro Translation Assay (Activation by Cancer ProteasesCathepsin B or MMP-9

The general protocol for the rabbit retoculocyte lysate reaction to testthe cytotoxicity of cancer protease-activiated proricin is describedbriefly in Example 3 and is described in more detail in Example 2.

Results

Activation of pAP 214 and pAP 220 proricin variants by cathepsin B andMMP-9, based on the method of May et al. (EMBO J. 8:301-308, 1989), isillustrated in FIGS. 50 and 51 respectively. The appearance of the 390base pair product (positive control) is observed in Lane F of FIG. 50and Lane G of FIG. 51. This 390 base pair product is absent in thenegative control lanes. Without cathepsin or MMP-9 activation, no orminimal N-glycosidase activity in the pAP 214 variant (Lanes H to L,FIG. 50) or the pAP 220 variant (Lanes A to E, FIG. 51) was observed.When the pAP 214 variant and the pAP 220 variant were activated bycathepsin or MMP-9 respectively, appearance of the 390 base pair productwas observed in a proricin concentration-dependent manner (Lanes A to Eof FIG. 50 and Lanes H to L of FIG. 51). The present experimental seriesdemonstrated the successful and selective activation of proricinvariants by cancer-associated proteases.

Example 7

The general protocol for the rabbit retoculocyte lysate reaction isdescribed briefly in Example 3 and is described in more detail inExample 2, all of which compliments the description below.

Depurination of Rabbit Reticulocyte 28S Ribosomal RNA by Digested andUndigested Ricin Variants

Affinity-purified mutant proricin mutants which were previously digestedwith the disease-specific protease, were reduced with 5%2-mercaptoethanol then diluted to 100 ng, 14.2 ng,2.0 ng,291 pg, and41.7 pg with 1×ENDO buffer(25 mM Tris pH 7.6, 25 mM KCl, 5 mM MgCl₂) andincubated with rabbit reticulocyte lysate, untreated (Promega) for 30minutes at 30° C. To compare the digested with the undigested proricinvariant, the proricin in digestion buffer (according to the specificdigestion protocol) was treated in the same manner as the digestedsample. As a positive and negative control, long of ricin A chain and1×ENDO buffer consecutively, was incubated with rabbit reticulocytelysate, untreated, for 30 min at 30° C.

Aniline Cleavage of rRNA and Gel Fractionation

Total RNA was then extracted from reticulocyte lysate translationmixtures with Trizol reagent (Gibco-BRL) as per manufacturer'sinstructions. The RNA was incubated with 80 ul of 1M aniline (distilled)with 2.8M acetic acid for 3 min at 60° C. in the dark.Ethanol-precipitated RNA samples were dissolved in 20 ul of 50%formamide, 0.1×E buffer (3.6 mM Tris, 3 mM NaH₂PO₄, 0.2 mM EDTA), and0.05% xylene cyanol. 10 ul of this was heated to 70° C. for 2 minutes,loaded and electrophoresed in 1.2% agarose, 0.1×E buffer, and 50%formamide gel with RNA running buffer (0.1×E buffer, 0.2% SDS).

Results

Activation of pAP-248 proricin variant by HCMV; pAP-256 by HAV3Cprotease; pAP-270 by MMP-2 protease; pAP-288 by t-PA protease; pAP-294by human neutrophil elastase; pAP-296 by calpain; and pAP-222 by MMP-2is illustrated in FIGS. 52, 55, 59, 61, 63, 65, and 67 respectively. Theappearance of the 390 base pair product (deposit of control) is obvervedin lane L of FIGS. 53, 55, 61, 63, 65 and 67. The 390 base pair productis observed in lane A of FIGS. 59 (activation of pAP-270 by MMP-2). This390 base pair product is absent in the negative control lanes. Withoutthe specific protease activation, no or minimal activity is seen in thelanes which contained only the proricin variant without digestion (seelane A, B, C, D, and E of FIGS. 53, 55, 61, 63, 65, and 67). The sameobservation is made in connection with pAP-270 in FIG. 59, however, theundigested lanes appear as H, I, J, K and L. When the variant wasactivated by its respective protease, there is an appearance of the 390base pair product in a proricin concentration-dependent manner (seeLanes H, I, J, K and L of FIG. 53, 55, 61, 63, 65, and 67 and Lanes A,B, C, D, and E of FIG. 59). The present experimental series demonstratethe successful and selective activation of the identified proricinvariants by selective corresponding proteases.

Example 8 Procedure for Examining the Cytotoxicity of Ricin and RicinVariants on the COS-1 Cell Line

Cell Preparation

After washing with 1×PBS (0.137 M NaCl, 2.68 mM KCl, 8.10 mM Na₂HPO₄,1.47 mM KH₂PO₄), cells in log phase growth were removed from plates with1×trypsin/EDTA (Gibco/BRL). The cells were centrifuged at 1100 rpm for 3min, resuspended in Dulbecco's Modified Eagle Medium containing 10%FBSand 1×pen/strep, and then counted using a haemocytometer. They wereadjusted to a concentration of 5×10⁴ cells•ml⁻¹. One hundred microlitersper well of cells was added to wells 2B-2G through to wells 9B-9G of aFalcon 96 well tissue culture plate. A separate 96 well tissue cultureplate was used for each sample of Ricin or Ricin variant. The plateswere incubated at 37° C. with 5% CO₂ for 24 hours.

Toxin Preparation

The Ricin and Ricin variants were sterile filtered using a 0.22 μmfilter (Millipore). The concentration of the sterile samples were thenquantified by A₂₈₀ and confirmed by BCA measurements (Pierce). For thevariants digested with the protease in vitro, the digests were carriedout as described in the digestion procedure for each protease. Thedigests were then diluted in the 1000 ng•ml⁻¹ dilution and sterilefiltered. The Ricin and the undigested pAP214 in the pAP 214cytotoxicity data were treated in the same manner but without theCathepsin B treatment. Ricin and Ricin variants were serially diluted tothe following concentrations: 1000 ng•ml⁻¹, 100 ng•ml⁻¹, 10 ng•ml⁻¹, 1ng•ml⁻¹, 0.1 ng•ml⁻¹, 0.01 ng•ml⁻¹, 0.001 ng•ml⁻¹ with media containing10%FBS and 1×pen/strep.

Application of Toxin or Variants to Plates

Columns 2 to 9 were labeled: control, 1000 ng•ml⁻¹, 100 ng•ml⁻¹, 10ng•ml⁻¹, 1 ng•ml⁻¹, 0.1 ng•ml⁻¹, 0.01 ng•ml⁻¹, 0.001 ng•ml⁻¹consecutively. The media was removed from all the sample wells with amultichannel pipettor. For each plate of variant and toxin, 50 μl ofmedia was added to wells 2B to 2G as the control, and 50 μl of eachsample dilution was added to the corresponding columns. For thepAP220+MMP-9 data, the plates were incubated for one hour at 37° C. with5% CO₂, then washed once and replaced with media, then incubated for 48hours at 37° C. with 5% CO₂. For the pAP 214+Cathepsin B data, the toxinwas left on the plates and incubated for 24 hours at 37° C. with 5% CO₂,then 50 μl of media was added to the wells with the toxin and incubatedfor another 24 hours at 37° C. with 5% CO₂.

Sample Application

The whole amount of media (and/or toxin)was removed from each well witha multichannel pipettor, and replaced with 100 μl of the substratemixture (Promega Cell Titer 96 Aqueous Non-Radioactive CellProliferation Assay Kit). The plates were incubated at 37° C. with 5%CO₂ for 2 to 4 hours, and subsequently read with a Spectramax 340 96well plate reader at 490 nm. The IC₅₀ values were calculated using theGRAFIT software program.

Results

In experiments with pAP-214 and Cathepsin B incubated with COS-1 cells,it may be seen that cells incubated with pAP-214 alone, pAP-214 wasineffective at causing cell death (see FIG. 56). However, thecytotoxicity of pAP-214 digested with Cathepsin B behaves similarly tothe ricin control in COS-1 cells. This is also illustrated in FIG. 56.Similarily, the cytotoxicity of undigested pAP-220 when incubated withCOS-1 cells is lower than the cytotoxicity observed with COS-1 cellsincubated with pAP-220 digested with MMP-9. Indeed the results suggestthat the toxicity of digested pAP-220 is greater than that of ricin.(See FIG. 57).

Example 9 Procedure for Examining the Cytotoxicity of Ricin and RicinVariants on Various Tissue Culture Cell Lines

Cell Preparation

After washing with 1×PBS (1.37M NaCl, 26.8 mM KCl, 81 mM Na₂HPO₄, 14.7mM KH₂PO₄), cells in log phase growth were removed from plates with1×trypsin/EDTA (Gibco/BRL). The cells were centrifuged at 1100 rpm for 3min, resuspended in media containing 10%FBS and 1×pen/strep (media useddepended on the cell line being tested), and then counted using ahaemocytometer. They were adjusted to a concentration of 5×10⁴cells•ml⁻¹ (faster growing cell lines were adjusted to 2×10⁴cells•ml⁻¹). One hundred microliters per well of cells was added towells 2B-2G through to wells 9B-9G of a Falcon 96 well tissue cultureplate. A separate 96 well tissue culture plate was used for each sampleof Ricin or Ricin variant. The plates were incubated at 37° C. with 5%CO₂ for 24 hours.

Toxin Preparation

The Ricin and Ricin variants were sterile filtered using a 0.22 μmfilter (Millipore). The concentration of the sterile samples were thenquantified by A₂₈₀ and confirmed by a BCA measurement (Pierce). Ricinand Ricin variants were serially diluted to the followingconcentrations: 3000 ng•ml⁻¹, 300 ng•ml⁻¹, 30 ng•ml⁻¹, 3 ng•ml⁻¹, 0.3ng•ml⁻¹, 0.03 ng•ml⁻¹, 0.003 ng•ml⁻¹ with media containing 10%FBS and1×pen/strep.

Application of Toxin or Variants to Plates

Columns 2 to 9 were labeled: control, 0.001 ng•ml⁻¹, 0.01 ng•ml⁻¹, 0.1ng•ml⁻¹, 1 ng•ml⁻¹, 10 ng•ml⁻¹, 100 ng•ml⁻¹, 1000 ng•ml⁻¹ consecutively.For each plate of variant and toxin, 50 μl of media was added to wells2B to 2G as the control, and 50 μl of each sample dilution was added tothe corresponding columns containing 100 μl per well of cells (i.e. 50μl of the 3000 ng•ml dilution added to the wells B-G in column 9,labeled 1000 ng•ml⁻¹). The plates were incubated for 48 hours at 37° C.with 5% CO₂.

Sample Application

An amount of 140 μl was removed from each well with a multichannelpipettor, and replaced with 100 μl of the substrate mixture (PromegaCell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit). Theplates were incubated at 37° C. with 5% CO₂ for 2 to 4 hours, andsubsequently read with a Spectramax 340 96 well plate reader at 490 nm.The IC₅₀ values were calculated using the GRAFIT software program.

Results

Referring to Table 2, it may be seen that the survival of cells iscorrelated with the proricin variant and the cell specific proteaseproduced by the cell type. For example, in the HT1080 cell line, bothpAP-214 and pAP-220 required only 2½ times the amount of ricin toachieve the same level of cytotoxicity. On the other hand, pAP-224required 193 times the amount of ricin to achieve the same level of celldeath. As well, it may be seen that in the cells where expression ofCathepsin D is found, pAP-214 and 220 were more effective at causingcell death than ricin and more effective than pAP-224. Detailsconcerning the various cells types used in these experiments areoutlined below.

COS-1 (African Green Monkey Kidney Cells)

This is an SV40 transformed cell line which was prepared fromestablished simian cells CV-1. (Reference: Gluzman, Y. (1975) Cell, 23,175-182)(ATCC CRL 1650)

HT-1080 Human Fibrosarcoma

(ATCC CCL 121) This cell line was shown to produce active MMP-9 intissue culture. References: Moore et al. (1997) Gynecologic Oncology 65,83-88.

9L Rat Glioblastoma

Glioblastomas are generally associated with cathepsin B expression.Levels of cathepsin B expression correspond to the extent of progressionof malignancy i.e. highest levels for glioblastomas over anaplasticastrocytomas over low-grade gliomas and normal brain tissue. The 9L cellline was provided by Dr. William Jia of the B. C. Cancer Agency.References: Mikkelsen et al. (Aug. 1995) Journal of Neurosurgery 83(2),285-290. Nakano et al. (1995) J. of Neurosurgery 83(2), 298-307.

MCF-7 Human Breast Cancer Cell Line (Epithilial)

(ATCC CRL 1555) In the absence of estrogen cathepsin B has not beenshown to be elevated relative to normal cells. It can be induced withestrogen to produce Cathepsin D. Production of MMP-9 is unknown.

Having illustrated and described the principles of the invention in apreferred embodiment, it should be appreciated to those skilled in theart that the invention can be modified in arrangement and detail withoutdeparture from such principles. We claim all modifications coming withinthe scope of the following claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

FULL CITATIONS FOR CERTAIN REFERENCES REFERRED TO IN THE SPECIFICATION

Bever Jr., C. T., Panitch, H. S., and Johnson, K. P. (1994) Neurology44(4), 745-8. Increased cathepsin B activity in peripheral bloodmononuclear cells of multiple sclerosis patients.

Cohen, P., Graves, H. C., Peehl, D. M., Kamarei, M., Giudice, L. C., andRosenfeld, R. G. (1992) Journal of Clinal Endocrinology and Metabolism75(4), 1046-53. Prostate-specific antigen (PSA) is an insulin-likegrowth factor binding protein-3 protease found in seminal plasma.

Conover, C. A. and De Leon, D. D. (1994) J. Biol. Chem. 269(10),7076-80. Acid activated insulin-like growth factor-binding protein-3proteolysis in normal and transformed cells. Role of cathepsin D.

Hansen, G., Schuster, A., Zubrod, C., and Wahn, V. (1995) Respiration62(3), 117-24. Alpha 1-proteinase inhibitor abrogates proteolytic andsecretagogue activity of cystic fibrosis sputum.

Muller, H. L., Oh, Y., Gargosky, S. E., Lehrnbecher, T., Hintz, R. L.,and Rosenfeld, R. G. (1993) Journal of Clinical Endocrinology andMetabolism 77(5), 1113-9. Concentrations of insulin-like growth factor(IGF)-binding protein-3 (IGFBP-3), IGF, and IGFBP-3 protease activity incerebrospinal fluid of children with leukemia, central nervous systemtumor, or meningitis.

TABLE 1 Table I - Sequence and Location of Oligonucleotide PrimersCorresponds to preproricin nucleotide Name of numbers: (see PrimerPrimer Sequence † FIGS. 8-10) Ricin-109 5′ -GGAGATGAAACCGGGAGGAAATACTATTGTAAT-3′ 27 to 59 Ricin-99Eco 5′ -GCGGAATTCCGGGAGGAAATACTATTGTAAT -3′ 37 to 59 Ricin267 5′ -ACGGTTTATTTTAGTTGA -3′ 300 to 317 Ricin486 5′ - ACTTGCTGGTAATCTGAG -3′519 to 536 Ricin725 5′ - AGAATAGTTGGGGGAGAC -3′ 758 to 775 Ricin937 5′ -AATGCTGATGTTTGTATG -3′ 970 to 987 Ricin1151 5′ - CGGGAGTCTATGTGATGA -3′1184 to 1201 Ricin1399 5′ - GCAAATAGTGGACAAGTA -3′ 1432 to 1449Ricin1627 5′ - GGATTGGTGTTAGATGTG -3′ 1660 to 1677 Ricin1729C 5′ -ATAACTTGCTGTCCTTCA -3′ 1864 to 1846 Ricin1729C 5′ -CGCTCTAGATAACTTGCTGTCCTTTCA 1864 to 1846 Xba † underlined sequencesinserted for subeloning purposes and not included in final preproricinsequences

TABLE 2 Comparative Toxicities to Selected Cell Lines of Ricin and RicinProvariants IC50_(Ricin) IC50_(pAP214) IC50_(pAP220) IC50_(pAP224) CellLine (ng/ml) IC50_(Ricin) IC50_(Ricin) IC50_(Ricin) Cos-1 0.1 17 22 150HT1080 0.5 2.46 2.14 193 9L 10.8 1.3 1.7 32.3 MCF-7 0.09 27.8 40 742(without estrogen)

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 142 <210> SEQ ID NO 1 <211>LENGTH: 9632 <212> TYPE: DNA <213> ORGANISM: Baculovirus transfer vectorpVL1393 <400> SEQUENCE: 1 aagctttact cgtaaagcga gttgaaggat catatttagttgcgtttatg agataagatt 60 gaaagcacgt gtaaaatgtt tcccgcgcgt tggcacaactatttacaatg cggccaagtt 120 ataaaagatt ctaatctgat atgttttaaa acacctttgcggcccgagtt gtttgcgtac 180 gtgactagcg aagaagatgt gtggaccgca gaacagatagtaaaacaaaa ccctagtatt 240 ggagcaataa tcgatttaac caacacgtct aaatattatgatggtgtgca ttttttgcgg 300 gcgggcctgt tatacaaaaa aattcaagta cctggccagactttgccgcc tgaaagcata 360 gttcaagaat ttattgacac ggtaaaagaa tttacagaaaagtgtcccgg catgttggtg 420 ggcgtgcact gcacacacgg tattaatcgc accggttacatggtgtgcag atatttaatg 480 cacaccctgg gtattgcgcc gcaggaagcc atagatagattcgaaaaagc cagaggtcac 540 aaaattgaaa gacaaaatta cgttcaagat ttattaatttaattaatatt atttgcattc 600 tttaacaaat actttatcct attttcaaat tgttgcgcttcttccagcga accaaaacta 660 tgcttcgctt gctccgttta gcttgtagcc gatcagtggcgttgttccaa tcgacggtag 720 gattaggccg gatattctcc accacaatgt tggcaacgttgatgttacgt ttatgctttt 780 ggttttccac gtacgtcttt tggccggtaa tagccgtaaacgtagtgccg tcgcgcgtca 840 cgcacaacac cggatgtttg cgcttgtccg cggggtattgaaccgcgcga tccgacaaat 900 ccaccacttt ggcaactaaa tcggtgacct gcgcgtcttttttctgcatt atttcgtctt 960 tcttttgcat ggtttcctgg aagccggtgt acatgcggtttagatcagtc atgacgcgcg 1020 tgacctgcaa atctttggcc tcgatctgct tgtccttgatggcaacgatg cgttcaataa 1080 actcttgttt tttaacaagt tcctcggttt tttgcgccaccaccgcttgc agcgcgtttg 1140 tgtgctcggt gaatgtcgca atcagcttag tcaccaactgtttgctctcc tcctcccgtt 1200 gtttgatcgc gggatcgtac ttgccggtgc agagcacttgaggaattact tcttctaaaa 1260 gccattcttg taattctatg gcgtaaggca atttggacttcataatcagc tgaatcacgc 1320 cggatttagt aatgagcact gtatgcggct gcaaatacagcgggtcgccc cttttcacga 1380 cgctgttaga ggtagggccc ccattttgga tggtctgctcaaataacgat ttgtatttat 1440 tgtctacatg aacacgtata gctttatcac aaactgtatattttaaactg ttagcgacgt 1500 ccttggccac gaaccggacc tgttggtcgc gctctagcacgtaccgcagg ttgaacgtat 1560 cttctccaaa tttaaattct ccaattttaa cgcgagccattttgatacac gtgtgtcgat 1620 tttgcaacaa ctattgtttt ttaacgcaaa ctaaacttattgtggtaagc aataattaaa 1680 tatgggggaa catgcgccgc tacaacactc gtcgttatgaacgcagacgg cgccggtctc 1740 ggcgcaagcg gctaaaacgt gttgcgcgtt caacgcggcaaacatcgcaa aagccaatag 1800 tacagttttg atttgcatat taacggcgat tttttaaattatcttattta ataaatagtt 1860 atgacgccta caactccccg cccgcgttga ctcgctgcacctcgagcagt tcgttgacgc 1920 cttcctccgt gtggccgaac acgtcgagcg ggtggtcgatgaccagcggc gtgccgcacg 1980 cgacgcacaa gtatctgtac accgaatgat cgtcgggcgaaggcacgtcg gcctccaagt 2040 ggcaatattg gcaaattcga aaatatatac agttgggttgtttgcgcata tctatcgtgg 2100 cgttgggcat gtacgtccga acgttgattt gcatgcaagccgaaattaaa tcattgcgat 2160 tagtgcgatt aaaacgttgt acatcctcgc ttttaatcatgccgtcgatt aaatcgcgca 2220 atcgagtcaa gtgatcaaag tgtggaataa tgttttctttgtattcccga gtcaagcgca 2280 gcgcgtattt taacaaacta gccatcttgt aagttagtttcatttaatgc aactttatcc 2340 aataatatat tatgtatcgc acgtcaagaa ttaacaatgcgcccgttgtc gcatctcaac 2400 acgactatga tagagatcaa ataaagcgcg aattaaatagcttgcgacgc aacgtgcacg 2460 atctgtgcac gcgttccggc acgagctttg attgtaataagtttttacga agcgatgaca 2520 tgacccccgt agtgacaacg atcacgccca aaagaactgccgactacaaa attaccgagt 2580 atgtcggtga cgttaaaact attaagccat ccaatcgaccgttagtcgaa tcaggaccgc 2640 tggtgcgaga agccgcgaag tatggcgaat gcatcgtataacgtgtggag tccgctcatt 2700 agagcgtcat gtttagacaa gaaagctaca tatttaattgatcccgatga ttttattgat 2760 aaattgaccc taactccata cacggtattc tacaatggcggggttttggt caaaatttcc 2820 ggactgcgat tgtacatgct gttaacggct ccgcccactattaatgaaat taaaaattcc 2880 aattttaaaa aacgcagcaa gagaaacatt tgtatgaaagaatgcgtaga aggaaagaaa 2940 aatgtcgtcg acatgctgaa caacaagatt aatatgcctccgtgtataaa aaaaatattg 3000 aacgatttga aagaaaacaa tgtaccgcgc ggcggtatgtacaggaagag gtttatacta 3060 aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtgaaaaccgatg tttaatcaag 3120 gctctgacgc atttctacaa ccacgactcc aagtgtgtgggtgaagtcat gcatctttta 3180 atcaaatccc aagatgtgta taaaccacca aactgccaaaaaatgaaaac tgtcgacaag 3240 ctctgtccgt ttgctggcaa ctgcaagggt ctcaatcctatttgtaatta ttgaataata 3300 aaacaattat aaatgctaaa tttgtttttt attaacgatacaaaccaaac gcaacaagaa 3360 catttgtagt attatctata attgaaaacg cgtagttataatcgctgagg taatatttaa 3420 aatcattttc aaatgattca cagttaattt gcgacaatataattttattt tcacataaac 3480 tagacgcctt gtcgtcttct tcttcgtatt ccttctctttttcatttttc tcctcataaa 3540 aattaacata gttattatcg tatccatata tgtatctatcgtatagagta aattttttgt 3600 tgtcataaat atatatgtct tttttaatgg ggtgtatagtaccgctgcgc atagtttttc 3660 tgtaatttac aacagtgcta ttttctggta gttcttcggagtgtgttgct ttaattatta 3720 aatttatata atcaatgaat ttgggatcgt cggttttgtacaatatgttg ccggcatagt 3780 acgcagcttc ttctagttca attacaccat tttttagcagcaccggatta acataacttt 3840 ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacctcccttttct atactattgt 3900 ctgcgagcag ttgtttgttg ttaaaaataa cagccattgtaatgagacgc acaaactaat 3960 atcacaaact ggaaatgtct atcaatatat agttgctgatatcatggaga taattaaaat 4020 gataaccatc tcgcaaataa ataagtattt tactgttttcgtaacagttt tgtaataaaa 4080 aaacctataa atattccgga ttattcatac cgtcccaccatcgggcgcgg atcccgggta 4140 ccttctagaa ttccggagcg gccgctgcag atctgatcctttcctgggac ccggcaagaa 4200 ccaaaaactc actctcttca aggaaatccg taatgttaaacccgacacga tgaagcttgt 4260 cgttggatgg aaaggaaaag agttctacag ggaaacttggacccgcttca tggaagacag 4320 cttccccatt gttaacgacc aagaagtgat ggatgttttccttgttgtca acatgcgtcc 4380 cactagaccc aaccgttgtt acaaattcct ggcccaacacgctctgcgtt gcgaccccga 4440 ctatgtacct catgacgtga ttaggatcgt cgagccttcatgggtgggca gcaacaacga 4500 gtaccgcatc agcctggcta agaagggcgg cggctgcccaataatgaacc ttcactctga 4560 gtacaccaac tcgttcgaac agttcatcga tcgtgtcatctgggagaact tctacaagcc 4620 catcgtttac atcggtaccg actctgctga agaggaggaaattctccttg aagtttccct 4680 ggtgttcaaa gtaaaggagt ttgcaccaga cgcacctctgttcactggtc cggcgtatta 4740 aaacacgata cattgttatt agtacattta ttaagcgctagattctgtgc gttgttgatt 4800 tacagacaat tgttgtacgt attttaataa ttcattaaatttataatctt tagggtggta 4860 tgttagagcg aaaatcaaat gattttcagc gtctttatatctgaatttaa atattaaatc 4920 ctcaatagat ttgtaaaata ggtttcgatt agtttcaaacaagggttgtt tttccgaacc 4980 gatggctgga ctatctaatg gattttcgct caacgccacaaaacttgcca aatcttgtag 5040 cagcaatcta gctttgtcga tattcgtttg tgttttgttttgtaataaag gttcgacgtc 5100 gttcaaaata ttatgcgctt ttgtatttct ttcatcactgtcgttagtgt acaattgact 5160 cgacgtaaac acgttaaata aagcttggac atatttaacatcgggcgtgt tagctttatt 5220 aggccgatta tcgtcgtcgt cccaaccctc gtcgttagaagttgcttccg aagacgattt 5280 tgccatagcc acacgacgcc tattaattgt gtcggctaacacgtccgcga tcaaatttgt 5340 agttgagctt tttggaatta tttctgattg cgggcgtttttgggcgggtt tcaatctaac 5400 tgtgcccgat tttaattcag acaacacgtt agaaagcgatggtgcaggcg gtggtaacat 5460 ttcagacggc aaatctacta atggcggcgg tggtggagctgatgataaat ctaccatcgg 5520 tggaggcgca ggcggggctg gcggcggagg cggaggcggaggtggtggcg gtgatgcaga 5580 cggcggttta ggctcaaatg tctctttagg caacacagtcggcacctcaa ctattgtact 5640 ggtttcgggc gccgtttttg gtttgaccgg tctgagacgagtgcgatttt tttcgtttct 5700 aatagcttcc aacaattgtt gtctgtcgtc taaaggtgcagcgggttgag gttccgtcgg 5760 cattggtgga gcgggcggca attcagacat cgatggtggtggtggtggtg gaggcgctgg 5820 aatgttaggc acgggagaag gtggtggcgg cggtgccgccggtataattt gttctggttt 5880 agtttgttcg cgcacgattg tgggcaccgg cgcaggcgccgctggctgca caacggaagg 5940 tcgtctgctt cgaggcagcg cttggggtgg tggcaattcaatattataat tggaatacaa 6000 atcgtaaaaa tctgctataa gcattgtaat ttcgctatcgtttaccgtgc cgatatttaa 6060 caaccgctca atgtaagcaa ttgtattgta aagagattgtctcaagctcg ccgcacgccg 6120 ataacaagcc ttttcatttt tactacagca ttgtagtggcgagacacttc gctgtcgtcg 6180 acgtacatgt atgctttgtt gtcaaaaacg tcgttggcaagctttaaaat atttaaaaga 6240 acatctctgt tcagcaccac tgtgttgtcg taaatgttgtttttgataat ttgcgcttcc 6300 gcagtatcga cacgttcaaa aaattgatgc gcatcaattttgttgttcct attattgaat 6360 aaataagatt gtacagattc atatctacga ttcgtcatggccaccacaaa tgctacgctg 6420 caaacgctgg tacaatttta cgaaaactgc aaaaacgtcaaaactcggta taaaataatc 6480 aacgggcgct ttggcaaaat atctatttta tcgcacaagcccactagcaa attgtatttg 6540 cagaaaacaa tttcggcgca caattttaac gctgacgaaataaaagttca ccagttaatg 6600 agcgaccacc caaattttat aaaaatctat tttaatcacggttccatcaa caaccaagtg 6660 atcgtgatgg actacattga ctgtcccgat ttatttgaaacactacaaat taaaggcgag 6720 ctttcgtacc aacttgttag caatattatt agacagctgtgtgaagcgct caacgatttg 6780 cacaagcaca atttcataca caacgacata aaactcgaaaatgtcttata tttcgaagca 6840 cttgatcgcg tgtatgtttg cgattacgga ttgtgcaaacacgaaaactc acttagcgtg 6900 cacgacggca cgttggagta ttttagtccg gaaaaaattcgacacacaac tatgcacgtt 6960 tcgtttgact ggtacgcggc gtgttaacat acaagttgctaacgtaatca tggtcatagc 7020 tgtttcctgt gtgaaattgt tatccgctca caattccacacaacatacga gccggaagca 7080 taaagtgtaa agcctggggt gcctaatgag tgagctaactcacattaatt gcgttgcgct 7140 cactgcccgc tttccagtcg ggaaacctgt cgtgccagctgcattaatga atcggccaac 7200 gcgcggggag aggcggtttg cgtattgggc gctcttccgcttcctcgctc actgactcgc 7260 tgcgctcggt cgttcggctg cggcgagcgg tatcagctcactcaaaggcg gtaatacggt 7320 tatccacaga atcaggggat aacgcaggaa agaacatgtgagcaaaaggc cagcaaaagg 7380 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttccataggctccgc ccccctgacg 7440 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaacccgacagga ctataaagat 7500 accaggcgtt tccccctgga agctccctcg tgcgctctcctgttccgacc ctgccgctta 7560 ccggatacct gtccgccttt ctcccttcgg gaagcgtggcgctttctcat agctcacgct 7620 gtaggtatct cagttcggtg taggtcgttc gctccaagctgggctgtgtg cacgaacccc 7680 ccgttcagcc cgaccgctgc gccttatccg gtaactatcgtcttgagtcc aacccggtaa 7740 gacacgactt atcgccactg gcagcagcca ctggtaacaggattagcaga gcgaggtatg 7800 taggcggtgc tacagagttc ttgaagtggt ggcctaactacggctacact agaaggacag 7860 tatttggtat ctgcgctctg ctgaagccag ttaccttcggaaaaagagtt ggtagctctt 7920 gatccggcaa acaaaccacc gctggtagcg gtggtttttttgtttgcaag cagcagatta 7980 cgcgcagaaa aaaaggatct caagaagatc ctttgatcttttctacgggg tctgacgctc 8040 agtggaacga aaactcacgt taagggattt tggtcatgagattatcaaaa aggatcttca 8100 cctagatcct tttaaattaa aaatgaagtt ttaaatcaatctaaagtata tatgagtaaa 8160 cttggtctga cagttaccaa tgcttaatca gtgaggcacctatctcagcg atctgtctat 8220 ttcgttcatc catagttgcc tgactccccg tcgtgtagataactacgata cgggagggct 8280 taccatctgg ccccagtgct gcaatgatac cgcgagacccacgctcaccg gctccagatt 8340 tatcagcaat aaaccagcca gccggaaggg ccgagcgcagaagtggtcct gcaactttat 8400 ccgcctccat ccagtctatt aattgttgcc gggaagctagagtaagtagt tcgccagtta 8460 atagtttgcg caacgttgtt gccattgcta caggcatcgtggtgtcacgc tcgtcgtttg 8520 gtatggcttc attcagctcc ggttcccaac gatcaaggcgagttacatga tcccccatgt 8580 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgttgtcagaagt aagttggccg 8640 cagtgttatc actcatggtt atggcagcac tgcataattctcttactgtc atgccatccg 8700 taagatgctt ttctgtgact ggtgagtact caaccaagtcattctgagaa tagtgtatgc 8760 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataataccgcgcca catagcagaa 8820 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcgaaaactctca aggatcttac 8880 cgctgttgag atccagttcg atgtaaccca ctcgtgcacccaactgatct tcagcatctt 8940 ttactttcac cagcgtttct gggtgagcaa aaacaggaaggcaaaatgcc gcaaaaaagg 9000 gaataagggc gacacggaaa tgttgaatac tcatactcttcctttttcaa tattattgaa 9060 gcatttatca gggttattgt ctcatgagcg gatacatatttgaatgtatt tagaaaaata 9120 aacaaatagg ggttccgcgc acatttcccc gaaaagtgccacctgacgtc taagaaacca 9180 ttattatcat gacattaacc tataaaaata ggcgtatcacgaggcccttt cgtctcgcgc 9240 gtttcggtga tgacggtgaa aacctctgac acatgcagctcccggagacg gtcacagctt 9300 gtctgtaagc ggatgccggg agcagacaag cccgtcagggcgcgtcagcg ggtgttggcg 9360 ggtgtcgggg ctggcttaac tatgcggcat cagagcagattgtactgaga gtgcaccata 9420 tgcggtgtga aataccgcac agatgcgtaa ggagaaaataccgcatcagg cgccattcgc 9480 cattcaggct gcgcaactgt tgggaagggc gatcggtgcgggcctcttcg ctattacgcc 9540 agctggcgaa agggggatgt gctgcaaggc gattaagttgggtaacgcca gggttttccc 9600 agtcacgacg ttgtaaaacg acggccagtg cc 9632<210> SEQ ID NO 2 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:Cathepsin B linker regions of pAP-213 <400> SEQUENCE: 2 tctttgcttaaatcgagaat ggtgccaaat tttaat 36 <210> SEQ ID NO 3 <211> LENGTH: 1855<212> TYPE: DNA <213> ORGANISM: Cathepsin B linker regions of pAP-214<400> SEQUENCE: 3 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt tttctttgct taaatcgaga atggtgccaa attttaatgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 4 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: MMP-3 linker regions of pAP-215 <400> SEQUENCE: 4 cgtccgaagccacagcaatt ttttggactt atgaat 36 <210> SEQ ID NO 5 <211> LENGTH: 1855<212> TYPE: DNA <213> ORGANISM: pAP-216 insert <400> SEQUENCE: 5gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900tcgtcacagt ttcgtccgaa gccacagcaa ttttttggac ttatgaatgc tgatgtttgt 960atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210>SEQ ID NO 6 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: MMP-7linker regions of pAP-217 <400> SEQUENCE: 6 tctttgcgtc cactggcattgtggcgaagt tttaat 36 <210> SEQ ID NO 7 <211> LENGTH: 1855 <212> TYPE:DNA <213> ORGANISM: pAP-218 insert <400> SEQUENCE: 7 gaattcatgaaaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttggatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacccaattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagagctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaaacagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcagagctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctggaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatcttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttgaacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggaggctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgttcctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggagaaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacacttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttgctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagtatattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagttttctttgcg tccactggca ttgtggcgaa gttttaatgc tgatgtttgt 960 atggatcctgagcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagattccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagctctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacgggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgccacccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttagcagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgttagtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctatatggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaaaggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgagataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtggccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgtatagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattctttaccctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattactctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgtaaattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 8<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: MMP-9 linker regions ofpAP-219 <400> SEQUENCE: 8 tctccgcaag gaattgcagg gcagcgaaat tttaat 36<210> SEQ ID NO 9 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM:pAP-220 insert <400> SEQUENCE: 9 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt tttctccgca aggaattgcagggcagcgaa attttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgtaggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaatacagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagacaatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctatgtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggataatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagtggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctactaataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagcaaatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggctctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgattctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaacgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagatgtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgacccaaaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcctgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggacagcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 10 <211> LENGTH: 42<212> TYPE: DNA <213> ORGANISM: THERM-MMP linker regions of pAP-221<400> SEQUENCE: 10 gatgtggatg aaagggatgt gagggaattt gcttcttttt ta 42<210> SEQ ID NO 11 <211> LENGTH: 1861 <212> TYPE: DNA <213> ORGANISM:pAP-222 insert <400> SEQUENCE: 11 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt ttgatgtgga tgaaagggatgtgagggaat ttgcttcttt tttagctgat 960 gtttgtatgg atcctgagcc catagtgcgtatcgtaggtc gaaatggtct atgtgttgat 1020 gttagggatg gaagattcca caacggaaacgcaatacagt tgtggccatg caagtctaat 1080 acagatgcaa atcagctctg gactttgaaaagagacaata ctattcgatc taatggaaag 1140 tgtttaacta cttacgggta cagtccgggagtctatgtga tgatctatga ttgcaatact 1200 gctgcaactg atgccacccg ctggcaaatatgggataatg gaaccatcat aaatcccaga 1260 tctagtctag ttttagcagc gacatcagggaacagtggta ccacacttac agtgcaaacc 1320 aacatttatg ccgttagtca aggttggcttcctactaata atacacaacc ttttgttaca 1380 accattgttg ggctatatgg tctgtgcttgcaagcaaata gtggacaagt atggatagag 1440 gactgtagca gtgaaaaggc tgaacaacagtgggctcttt atgcagatgg ttcaatacgt 1500 cctcagcaaa accgagataa ttgccttacaagtgattcta atatacggga aacagttgtt 1560 aagatcctct cttgtggccc tgcatcctctggccaacgat ggatgttcaa gaatgatgga 1620 accattttaa atttgtatag tggattggtgttagatgtga ggcgatcgga tccgagcctt 1680 aaacaaatca ttctttaccc tctccatggtgacccaaacc aaatatggtt accattattt 1740 tgatagacag attactctct tgcagtgtgtgtgtcctgcc atgaaaatag atggcttaaa 1800 taaaaaggac attgtaaatt ttgtaactgaaaggacagca agttatatcg aattcctgca 1860 g 1861 <210> SEQ ID NO 12 <211>LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: P.falciparum-A linker regionsof pAP-223 <400> SEQUENCE: 12 caggtggttc aattgcagaa ttatgatgaa gaggat 36<210> SEQ ID NO 13 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM:pAP-224 insert <400> SEQUENCE: 13 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt ttcaggtggt tcaattgcagaattatgatg aagaggatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgtaggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaatacagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagacaatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctatgtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggataatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagtggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctactaataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagcaaatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggctctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgattctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaacgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagatgtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgacccaaaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcctgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggacagcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 14 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: P.falciparum-B linker regions of pAP-225<400> SEQUENCE: 14 ttgccgattt tcggggaatc ggaggacaat gatgaa 36 <210> SEQID NO 15 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-226insert <400> SEQUENCE: 15 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt ttttgccgat tttcggggaa tcggaggacaatgatgaagc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 16 <211> LENGTH: 36 <212> TYPE:DNA <213> ORGANISM: P. falciparum-C linker regions of pAP-227 <400>SEQUENCE: 16 caggtggtta caggggaagc gatatcagtt actatg 36 <210> SEQ ID NO17 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-228 insert<400> SEQUENCE: 17 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt ttcaggtggt tacaggggaa gcgatatcagttactatggc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 18 <211> LENGTH: 36 <212> TYPE:DNA <213> ORGANISM: P. falciparum-D linker regions of pAP-229 <400>SEQUENCE: 18 gctttggaga gaacgttcct gtcgttccct actaat 36 <210> SEQ ID NO19 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-230 insert<400> SEQUENCE: 19 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt ttgctttgga gagaacgttc ctgtcgttccctactaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 20 <211> LENGTH: 36 <212> TYPE:DNA <213> ORGANISM: P. falciparum-E linker regions of pAP-231 <400>SEQUENCE: 20 aaattccaag atatgctaaa taattcacag catcag 36 <210> SEQ ID NO21 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-232 insert<400> SEQUENCE: 21 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt ttaaattcca agatatgcta aataattcacagcatcaggc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 22 <211> LENGTH: 36 <212> TYPE:DNA <213> ORGANISM: HSV-A linker regions of pAP-233 <400> SEQUENCE: 22tctgcgcttg taaacgcatc gtcggcacat gttaat 36 <210> SEQ ID NO 23 <211>LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-234 insert <400>SEQUENCE: 23 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt tttctgcgct tgtaaacgca tcgtcggcac atgttaatgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 24 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: HSV-B linker regions of pAP-235 <400> SEQUENCE: 24 tctacgtatttacaggcatc ggagaaattt aagaat 36 <210> SEQ ID NO 25 <211> LENGTH: 1855<212> TYPE: DNA <213> ORGANISM: pAP-236 insert <400> SEQUENCE: 25gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900tcgtcacagt tttctacgta tttacaggca tcggagaaat ttaagaatgc tgatgtttgt 960atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210>SEQ ID NO 26 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: VZV-Alinker regions of pAP-237 <400> SEQUENCE: 26 tctcaggatg taaacgcagtggaggcaagt tctaat 36 <210> SEQ ID NO 27 <211> LENGTH: 1855 <212> TYPE:DNA <213> ORGANISM: pAP-238 insert <400> SEQUENCE: 27 gaattcatgaaaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttggatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacccaattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagagctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaaacagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcagagctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctggaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatcttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttgaacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggaggctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgttcctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggagaaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacacttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttgctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagtatattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagttttctcagga tgtaaacgca gtggaggcaa gttctaatgc tgatgtttgt 960 atggatcctgagcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagattccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagctctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacgggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgccacccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttagcagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgttagtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctatatggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaaaggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgagataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtggccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgtatagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattctttaccctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattactctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgtaaattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO28 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: VZV-B linker regionsof pAP-239 <400> SEQUENCE: 28 tctgtgtatt tacaggcatc gacgggatat ggtaat 36<210> SEQ ID NO 29 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM:pAP-240 insert <400> SEQUENCE: 29 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt tttctgtgta tttacaggcatcgacgggat atggtaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgtaggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaatacagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagacaatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctatgtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggataatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagtggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctactaataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagcaaatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggctctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgattctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaacgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagatgtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgacccaaaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcctgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggacagcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 30 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: EBV-A linker regions of pAP-241 <400>SEQUENCE: 30 tctaagcttg tacaggcatc ggcgtcaggt gttaat 36 <210> SEQ ID NO31 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-242 insert<400> SEQUENCE: 31 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt ttgtttcgca gaactatcca atagtgcaaaattttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 32 <211> LENGTH: 36 <212> TYPE:DNA <213> ORGANISM: EBV-B linker regions of pAP-243 <400> SEQUENCE: 32tcttcgtatc taaaggcatc ggacgcacct gataat 36 <210> SEQ ID NO 33 <211>LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-244 insert <400>SEQUENCE: 33 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt tttcttcgta tctaaaggca tcggacgcac ctgataatgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 34 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: CMV-A linker regions of pAP-245 <400> SEQUENCE: 34 tctggggttgtaaatgcatc gtgtagactt gctaat 36 <210> SEQ ID NO 35 <211> LENGTH: 1855<212> TYPE: DNA <213> ORGANISM: pAP-246 insert <400> SEQUENCE: 35gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900tcgtcacagt tttctggggt tgtaaatgca tcgtgtagac ttgctaatgc tgatgtttgt 960atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210>SEQ ID NO 36 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: CMV-Blinker regions of pAP-247 <400> SEQUENCE: 36 tcttcgtatg taaaggcatcggtgtcacct gaaaat 36 <210> SEQ ID NO 37 <211> LENGTH: 1855 <212> TYPE:DNA <213> ORGANISM: pPA-248 insert <400> SEQUENCE: 37 gaattcatgaaaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttggatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacccaattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagagctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaaacagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcagagctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctggaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatcttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttgaacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggaggctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgttcctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggagaaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacacttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttgctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagtatattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagttttcttcgta tgtaaaggca tcggtgtcac ctgaaaatgc tgatgtttgt 960 atggatcctgagcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagattccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagctctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacgggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgccacccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttagcagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgttagtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctatatggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaaaggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgagataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtggccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgtatagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattctttaccctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattactctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgtaaattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO38 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: HHV-6 linker regionsof pAP-249 <400> SEQUENCE: 38 tcttcgattt taaatgcatc ggtgccaaat tttaat 36<210> SEQ ID NO 39 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM:pAP-250 insert <400> SEQUENCE: 39 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt tttcttcgat tttaaatgcatcggtgccaa attttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgtaggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaatacagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagacaatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctatgtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggataatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagtggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctactaataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagcaaatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggctctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgattctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaacgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagatgtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgacccaaaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcctgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggacagcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 40 <211> LENGTH: 12<212> TYPE: PRT <213> ORGANISM: Cancer protease-sensitive AA linkers inpAP-213 and pAP-214 <400> SEQUENCE: 40 Ser Leu Leu Lys Ser Arg Met ValPro Asn Phe Asn 1 5 10 <210> SEQ ID NO 41 <211> LENGTH: 12 <212> TYPE:PRT <213> ORGANISM: Cancer protease-sensitive AA linkers in pAP-215 andpAP-216 <400> SEQUENCE: 41 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu MetAsn 1 5 10 <210> SEQ ID NO 42 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Cancer protease-sensitive AA linkers in pAP-217 and pAP-218<400> SEQUENCE: 42 Ser Leu Arg Pro Leu Ala Leu Trp Arg Ser Phe Asn 1 510 <210> SEQ ID NO 43 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:Cancer protease-sensitive AA linkers in pAP-219 and pAP-220 <400>SEQUENCE: 43 Ser Pro Gln Gly Ile Ala Gly Gln Arg Asn Phe Asn 1 5 10<210> SEQ ID NO 44 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:Cancer protease-sensitive AA linkers in aAP-221 and pAP-222 <400>SEQUENCE: 44 Asp Val Asp Glu Arg Asp Val Arg Gly Phe Ala Ser Phe Leu 1 510 <210> SEQ ID NO 45 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:Cancer protease-sensitive linkers pAP-241 and pAP-242 <400> SEQUENCE: 45Ser Lys Leu Val Gln Ala Ser Ala Ser Gly Val Asn 1 5 10 <210> SEQ ID NO46 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Cancerprotease-sensitive linkers pAP243 and pAP-244 <400> SEQUENCE: 46 Ser SerTyr Leu Lys Ala Ser Asp Ala Pro Asp Asn 1 5 10 <210> SEQ ID NO 47 <211>LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: ILV linker regions of pAP-253<400> SEQUENCE: 47 tctaagtatc tacaggcaaa tgaggtaatt actaat 36 <210> SEQID NO 48 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-254insert <400> SEQUENCE: 48 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt tttctaagta tctacaggca aatgaggtaattactaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 49 <211> LENGTH: 36 <212> TYPE:DNA <213> ORGANISM: HAV-A linker regions of pAP-257 <400> SEQUENCE: 49tctgagctta gaacgcaatc gttctcaaat tggaat 36 <210> SEQ ID NO 50 <211>LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-258 insert <400>SEQUENCE: 50 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt tttctgagct tagaacgcaa tcgttctcaa attggaatgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 51 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: HAV-B linker regions of pAP-255 <400> SEQUENCE: 51 tctgagctttggtcgcaagg gatcgatgat gataat 36 <210> SEQ ID NO 52 <211> LENGTH: 1855<212> TYPE: DNA <213> ORGANISM: pAP-256 insert <400> SEQUENCE: 52gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900tcgtcacagt tttctgagct ttggtcgcaa gggatcgatg atgataatgc tgatgtttgt 960atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210>SEQ ID NO 53 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: CAN linkerregions of pAP-259 <400> SEQUENCE: 53 tctaagcctg caaagttctt caggctaaattttaat 36 <210> SEQ ID NO 54 <211> LENGTH: 1855 <212> TYPE: DNA <213>ORGANISM: pAP-260 insert <400> SEQUENCE: 54 gaattcatga aaccgggaggaaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctcagggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaactttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcggtcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttggtttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgttacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgcatatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactgatgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgctggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagcgctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataatttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcacgagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatagttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaattcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccctatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt tttctaagcctgcaaagttc ttcaggctaa attttaatgc tgatgtttgt 960 atggatcctg agcccatagtgcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacggaaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggactttgaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtccgggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggcaaatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatcagggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttggcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtgcttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaacaacagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgccttacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatcctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggattggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctccatggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagtgtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaactgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 55 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: P.falciparumprotease-sensitive linkers pAP-223 and pAP-224 <400> SEQUENCE: 55 GlnVal Val Gln Leu Gln Asn Tyr Asp Glu Glu Asp 1 5 10 <210> SEQ ID NO 56<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: P.falciparumprotease-sensitive linkers pAP-225 and pAP-226 <400> SEQUENCE: 56 LeuPro Ile Phe Gly Glu Ser Glu Asp Asn Asp Glu 1 5 10 <210> SEQ ID NO 57<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: P.falciparumprotease-sensitive linker pAP-227 and pAP-228 <400> SEQUENCE: 57 Gln ValVal Thr Gly Glu Ala Ile Ser Val Thr Met 1 5 10 <210> SEQ ID NO 58 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: P.falciparumprotease-sensitive linkers pAP-229 and pAP-230 <400> SEQUENCE: 58 AlaLeu Glu Arg Thr Phe Leu Ser Phe Pro Thr Asn 1 5 10 <210> SEQ ID NO 59<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: P.falciparumprotease-sensitive linkers pAP-231 and pAP-232 <400> SEQUENCE: 59 LysPhe Gln Asp Met Leu Asn Ile Ser Gln His Gln 1 5 10 <210> SEQ ID NO 60<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-233 and pAP-234 <400> SEQUENCE: 60 SerAla Leu Val Asn Ala Ser Ser Ala His Val Asn 1 5 10 <210> SEQ ID NO 61<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-235 and pAP-236 <400> SEQUENCE: 61 SerThr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Asn 1 5 10 <210> SEQ ID NO 62<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-249 and pAP-250 <400> SEQUENCE: 62 SerSer Ile Leu Asn Ala Ser Val Pro Asn Phe Asn 1 5 10 <210> SEQ ID NO 63<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-245 and pAP-246 <400> SEQUENCE: 63 SerGly Val Val Asn Ala Ser Cys Arg Leu Ala Asn 1 5 10 <210> SEQ ID NO 64<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-247 and pAP-248 <400> SEQUENCE: 64 SerSer Tyr Val Lys Ala Ser Val Ser Pro Glu Asn 1 5 10 <210> SEQ ID NO 65<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-237 and aAP-238 <400> SEQUENCE: 65 SerGln Asp Val Asn Ala Val Glu Ala Ser Ser Asn 1 5 10 <210> SEQ ID NO 66<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-239 and pAP-240 <400> SEQUENCE: 66 SerVal Tyr Leu Gln Ala Ser Thr Gly Tyr Gly Asn 1 5 10 <210> SEQ ID NO 67<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-253 and pAP-254 <400> SEQUENCE: 67 SerLys Tyr Leu Gln Ala Asn Glu Val Ile Thr Asn 1 5 10 <210> SEQ ID NO 68<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-255 and pAP-256 <400> SEQUENCE: 68 SerGlu Leu Arg Thr Gln Ser Phe Ser Asn Trp Asn 1 5 10 <210> SEQ ID NO 69<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Viralprotease-sensitive linkers pAP-257 and pAP-258 <400> SEQUENCE: 69 SerGlu Leu Trp Ser Gln Gly Ile Asp Asp Asp Asn 1 5 10 <210> SEQ ID NO 70<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Candida asparticprotease-sensitive linkers pAP-259 and pAP- <400> SEQUENCE: 70 Ser LysPro Ala Lys Phe Phe Arg Leu Asn Phe Asn 1 5 10 <210> SEQ ID NO 71 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Candida asparticprotease-sensitive linkers pAP-261 and pAP- <400> SEQUENCE: 71 Ser LysPro Ile Glu Phe Phe Arg Leu Asn Phe Asn 1 5 10 <210> SEQ ID NO 72 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Candida asparticprotease-sensitive linkers pAP-263 and pAP- <400> SEQUENCE: 72 Ser LysPro Ala Glu Phe Phe Ala Leu Asn Phe Asn 1 5 10 <210> SEQ ID NO 73 <211>LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: HCV-A linker region ofpAP-262 <400> SEQUENCE: 73 gatttggagg tagtgacatc gacatgggtt tttaat 36<210> SEQ ID NO 74 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM:pAP-262 insert <400> SEQUENCE: 74 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt ttgatttgga ggtagtgacatcgacatggg tttttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgtaggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaatacagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagacaatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctatgtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggataatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagtggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctactaataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagcaaatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggctctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgattctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaacgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagatgtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgacccaaaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcctgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggacagcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 75 <211> LENGTH: 12<212> TYPE: PRT <213> ORGANISM: Mutant Preproricin linker region forHCV-A, pAP-262 <400> SEQUENCE: 75 Asp Leu Glu Val Val Thr Ser Thr TrpVal Phe Asn 1 5 10 <210> SEQ ID NO 76 <211> LENGTH: 36 <212> TYPE: DNA<213> ORGANISM: HCV-B linker region of pAP-264 <400> SEQUENCE: 76gatgagatgg aagagtgtgc gtcacacctt tttaat 36 <210> SEQ ID NO 77 <211>LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-264 insert <400>SEQUENCE: 77 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt ttgatgagat ggaagagtgt gcgtcacacc tttttaatgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 78 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Mutant preproricin linker region for HCV-B, pAP-264 <400>SEQUENCE: 78 Asp Glu Met Glu Glu Cys Ala Ser His Leu Phe Asn 1 5 10<210> SEQ ID NO 79 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:HCV-C linker region of pAP-266 <400> SEQUENCE: 79 gaggacgttg tatgttgttcgatgtcatat tttaat 36 <210> SEQ ID NO 80 <211> LENGTH: 1855 <212> TYPE:DNA <213> ORGANISM: pAP-266 insert <400> SEQUENCE: 80 gaattcatgaaaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttggatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacccaattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagagctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaaacagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcagagctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctggaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatcttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttgaacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggaggctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgttcctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggagaaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacacttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttgctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagtatattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagtttgaggacgt tgtatgttgt tcgatgtcat attttaatgc tgatgtttgt 960 atggatcctgagcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagattccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagctctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacgggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgccacccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttagcagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgttagtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctatatggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaaaggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgagataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtggccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgtatagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattctttaccctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattactctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgtaaattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO81 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Mutant preproricinlinker region for HCV-C, pAP-266 <400> SEQUENCE: 81 Glu Asp Val Val CysCys Ser Met Ser Tyr Phe Asn 1 5 10 <210> SEQ ID NO 82 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: HCV-D linker region of pAP-268 <400>SEQUENCE: 82 aaggggtgga gattgctagc gccaataact gcttat 36 <210> SEQ ID NO83 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-268 insert<400> SEQUENCE: 83 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt ttaaggggtg gagattgcta gcgccaataactgcttatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 84 <211> LENGTH: 12 <212> TYPE:PRT <213> ORGANISM: Mutant preproricin linker region for HCV-D, pAP-268<400> SEQUENCE: 84 Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr 1 510 <210> SEQ ID NO 85 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:MMP-2 linker region of pAP-270 <400> SEQUENCE: 85 tctttgcccc tgggtttatgggctcctaat tttaat 36 <210> SEQ ID NO 86 <211> LENGTH: 1855 <212> TYPE:DNA <213> ORGANISM: pAP-270 insert <400> SEQUENCE: 86 gaattcatgaaaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttggatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacccaattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagagctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaaacagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcagagctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctggaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatcttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttgaacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggaggctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgttcctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggagaaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacacttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttgctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagtatattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagttttctttgcc cctgggttta tgggctccta attttaatgc tgatgtttgt 960 atggatcctgagcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagattccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagctctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacgggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgccacccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttagcagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgttagtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctatatggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaaaggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgagataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtggccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgtatagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattctttaccctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattactctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgtaaattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO87 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Mutant preproricinlinker region for MMP-2, pAP-270 <400> SEQUENCE: 87 Ser Leu Pro Leu GlyLeu Trp Ala Pro Asn Phe Asn 1 5 10 <210> SEQ ID NO 88 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: Cathepsin B (Site 2) linker region ofpAP-272 <400> SEQUENCE: 88 tctttgctta tagctagaag gatgcctaat tttaat 36<210> SEQ ID NO 89 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM:pAP-272 insert <400> SEQUENCE: 89 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt tttctttgct tatagctagaaggatgccta attttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgtaggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaatacagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagacaatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctatgtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggataatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagtggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctactaataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagcaaatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggctctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgattctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaacgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagatgtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgacccaaaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcctgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggacagcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 90 <211> LENGTH: 12<212> TYPE: PRT <213> ORGANISM: Mutant preproricin linker region forCathepsin B (site 2), p <400> SEQUENCE: 90 Ser Leu Leu Ile Ala Arg ArgMet Pro Asn Phe Asn 1 5 10 <210> SEQ ID NO 91 <211> LENGTH: 36 <212>TYPE: DNA <213> ORGANISM: Cathepsin L linker region of pAP-274 <400>SEQUENCE: 91 tctttgctta tattccggtc atgggctaat tttaat 36 <210> SEQ ID NO92 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-274 insert<400> SEQUENCE: 92 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt tttctttgct tatattccgg tcatgggctaattttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 93 <211> LENGTH: 12 <212> TYPE:PRT <213> ORGANISM: Mutant preproricin linker region of Cathepsin L,pAP-274 <400> SEQUENCE: 93 Ser Leu Leu Ile Phe Arg Ser Trp Ala Asn PheAsn 1 5 10 <210> SEQ ID NO 94 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Cathepsin D linker region of pAP-276 <400> SEQUENCE: 94tctggtgttg tcatcgctac tgttattgtt atcacc 36 <210> SEQ ID NO 95 <211>LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-276 insert <400>SEQUENCE: 95 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt tttctggtgt tgtcatcgct actgttattg ttatcaccgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 96 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Mutant preproricin linker region for Cathepsin D, pAP-276<400> SEQUENCE: 96 Ser Gly Val Val Ile Ala Thr Val Ile Val Ile Thr 1 510 <210> SEQ ID NO 97 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:MMP-1 linker region of pAP-278 <400> SEQUENCE: 97 tctttgggtc ctcaaggcatttggggacag tttaat 36 <210> SEQ ID NO 98 <211> LENGTH: 1855 <212> TYPE:DNA <213> ORGANISM: pAP-278 insert <400> SEQUENCE: 98 gaattcatgaaaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttggatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacccaattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagagctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaaacagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcagagctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctggaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatcttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttgaacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggaggctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgttcctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggagaaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacacttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttgctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagtatattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagttttctttggg tcctcaaggc atttggggac agtttaatgc tgatgtttgt 960 atggatcctgagcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagattccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagctctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacgggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgccacccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttagcagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgttagtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctatatggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaaaggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgagataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtggccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgtatagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattctttaccctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattactctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgtaaattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO99 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Mutant preproricinlinker region for MMP-1, pAP-278 <400> SEQUENCE: 99 Ser Leu Gly Pro GlnGly Ile Trp Gly Gln Phe Asn 1 5 10 <210> SEQ ID NO 100 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: Urokinase-Type Plasminogen Activatorlinder region of pAP-28 <400> SEQUENCE: 100 aaaaaatccc ctggaagagttgtcggtggc tctgta 36 <210> SEQ ID NO 101 <211> LENGTH: 1855 <212> TYPE:DNA <213> ORGANISM: pAP-280 insert <400> SEQUENCE: 101 gaattcatgaaaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttggatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120 aaacaatacccaattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagagctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240 gtgttgccaaacagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300 aatcatgcagagctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctggaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420 atcactcatcttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480 gatagacttgaacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540 ctagaggaggctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600 ctggctcgttcctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660 attgagggagaaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720 gtaattacacttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780 ggagcctttgctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagtatattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900 tcgtcacagtttaaaaaatc ccctggaaga gttgtcggtg gctctgtagc tgatgtttgt 960 atggatcctgagcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagattccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080 gcaaatcagctctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140 actacttacgggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200 actgatgccacccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260 ctagttttagcagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320 tatgccgttagtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380 gttgggctatatggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440 agcagtgaaaaggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500 caaaaccgagataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtggccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgtatagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680 atcattctttaccctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740 acagattactctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800 ggacattgtaaattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO102 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Mutant preproricinlinker region for Urokinase-Type Plasmino <400> SEQUENCE: 102 Lys LysSer Pro Gly Arg Val Val Gly Gly Ser Val 1 5 10 <210> SEQ ID NO 103 <211>LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: MT-MMP linker region ofpAP-282 <400> SEQUENCE: 103 ccccaaggac tcctaggggc tcctggtatt cttggc 36<210> SEQ ID NO 104 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM:pAP-282 insert <400> SEQUENCE: 104 gaattcatga aaccgggagg aaatactattgtaatatgga tgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtctttcacattag aggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccacagcgggtgcca ctgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaacaactggagctg atgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctataaaccaacggt ttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcgctggatgtca ccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttctttcatcctgaca atcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaatcgatatacat tcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctgagagaaaata tcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattattacagtactg gtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaaatgatttcag aagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattaggtacaaccgga gatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggagactttccactg caattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaaagacgtaatg gttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagctctcatggtgt atagatgcgc acctccacca 900 tcgtcacagt ttccccaagg actcctaggggctcctggta ttcttggcgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgtaggtcgaaatg gtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaatacagttgtggc catgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagacaatactattc gatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctatgtgatgatct atgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggataatggaacca tcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagtggtaccacac ttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctactaataatacac aaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagcaaatagtggac aagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggctctttatgcag atggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgattctaatatac gggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaacgatggatgt tcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagatgtgaggcgat cggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgacccaaaccaaatat ggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcctgccatgaaa atagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggacagcaagttat atcgaattcc tgcag 1855 <210> SEQ ID NO 105 <211> LENGTH: 12<212> TYPE: PRT <213> ORGANISM: Mutant preproricin linker region forMT-MMP, PAP-282 <400> SEQUENCE: 105 Pro Gln Gly Leu Leu Gly Ala Pro GlyIle Leu Gly 1 5 10 <210> SEQ ID NO 106 <211> LENGTH: 93 <212> TYPE: DNA<213> ORGANISM: MMP-11 linker region of pAP-284 <400> SEQUENCE: 106cacggccccg agggtttaag agtgggattt tatgaatctg acgtcatggg aagaggccat 60gctcgtttag ttcatgtcga agagcctcac act 93 <210> SEQ ID NO 107 <211>LENGTH: 1912 <212> TYPE: DNA <213> ORGANISM: pAP-284 insert <400>SEQUENCE: 107 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt ttcacggccc cgagggttta agagtgggat tttatgaatctgacgtcatg 960 ggaagaggcc atgctcgttt agttcatgtc gaagagcctc acactgctgatgtttgtatg 1020 gatcctgagc ccatagtgcg tatcgtaggt cgaaatggtc tatgtgttgatgttagggat 1080 ggaagattcc acaacggaaa cgcaatacag ttgtggccat gcaagtctaatacagatgca 1140 aatcagctct ggactttgaa aagagacaat actattcgat ctaatggaaagtgtttaact 1200 acttacgggt acagtccggg agtctatgtg atgatctatg attgcaatactgctgcaact 1260 gatgccaccc gctggcaaat atgggataat ggaaccatca taaatcccagatctagtcta 1320 gttttagcag cgacatcagg gaacagtggt accacactta cagtgcaaaccaacatttat 1380 gccgttagtc aaggttggct tcctactaat aatacacaac cttttgttacaaccattgtt 1440 gggctatatg gtctgtgctt gcaagcaaat agtggacaag tatggatagaggactgtagc 1500 agtgaaaagg ctgaacaaca gtgggctctt tatgcagatg gttcaatacgtcctcagcaa 1560 aaccgagata attgccttac aagtgattct aatatacggg aaacagttgttaagatcctc 1620 tcttgtggcc ctgcatcctc tggccaacga tggatgttca agaatgatggaaccatttta 1680 aatttgtata gtggattggt gttagatgtg aggcgatcgg atccgagccttaaacaaatc 1740 attctttacc ctctccatgg tgacccaaac caaatatggt taccattattttgatagaca 1800 gattactctc ttgcagtgtg tgtgtcctgc catgaaaata gatggcttaaataaaaagga 1860 cattgtaaat tttgtaactg aaaggacagc aagttatatc gaattcctgcag 1912 <210> SEQ ID NO 108 <211> LENGTH: 31 <212> TYPE: PRT <213>ORGANISM: Mutant preproricin linker region for MMP-11, pAP-284 <400>SEQUENCE: 108 His Gly Pro Glu Gly Leu Arg Val Gly Phe Tyr Glu Ser AspVal Met 1 5 10 15 Gly Arg Gly His Ala Arg Leu Val His Val Glu Glu ProHis Thr 20 25 30 <210> SEQ ID NO 109 <211> LENGTH: 36 <212> TYPE: DNA<213> ORGANISM: MMP-13 linker region of pAP-286 <400> SEQUENCE: 109ggacctcagg ggcttgctgg tcaacgaggc attgtc 36 <210> SEQ ID NO 110 <211>LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-286 insert <400>SEQUENCE: 110 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt ttggacctca ggggcttgct ggtcaacgag gcattgtcgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 111 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Mutant preproricin linker region for MMP-13, pAP-286 <400>SEQUENCE: 111 Gly Pro Gln Gly Leu Ala Gly Gln Arg Gly Ile Val 1 5 10<210> SEQ ID NO 112 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:Tissue-type Plasminogen Activator linker region of pAP-288 <400>SEQUENCE: 112 ggcggatctg ggcaaagggg tcgtaaagct cttgaa 36 <210> SEQ ID NO113 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-288 insert<400> SEQUENCE: 113 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt ttggcggatc tgggcaaagg ggtcgtaaagctcttgaagc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 114 <211> LENGTH: 12 <212> TYPE:PRT <213> ORGANISM: Mutant preproricin linker region for Tissue-typePlasminogen <400> SEQUENCE: 114 Gly Gly Ser Gly Gln Arg Gly Arg Lys AlaLeu Glu 1 5 10 <210> SEQ ID NO 115 <211> LENGTH: 36 <212> TYPE: DNA<213> ORGANISM: Human Prostate-Specific Antigen linker region of pAP-290<400> SEQUENCE: 115 tctttgtcag ctcttctctc ttccgatatt tttaat 36 <210> SEQID NO 116 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-290insert <400> SEQUENCE: 116 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt tttctttgtc agctcttctc tcttccgatatttttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 117 <211> LENGTH: 12 <212> TYPE:PRT <213> ORGANISM: Mutant preproricin linker region-humanProstate-Specific Ant <400> SEQUENCE: 117 Ser Leu Ser Ala Leu Leu SerSer Asp Ile Phe Asn 1 5 10 <210> SEQ ID NO 118 <211> LENGTH: 36 <212>TYPE: DNA <213> ORGANISM: Kallikrein linker region of pAP-292 <400>SEQUENCE: 118 tctttgccta gatttaaaat tatcggtggc tttaat 36 <210> SEQ ID NO119 <211> LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-292 insert<400> SEQUENCE: 119 gaattcatga aaccgggagg aaatactatt gtaatatggatgtatgcagt ggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattagaggataacaa catattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgccactgtgcaaag ctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctgatgtgagaca tgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggtttattttagt tgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtcaccaatgcata tgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgacaatcaggaaga tgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacattcgcctttgg tggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaatatcgagttggg aaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactggtggcactca gcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcagaagcagcaag attccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccggagatctgcacc agatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactgcaattcaaga gtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatggttccaaatt cagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgtatagatgcgc acctccacca 900 tcgtcacagt tttctttgcc tagatttaaa attatcggtggctttaatgc tgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatggtctatgtgt tgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggccatgcaagtc taatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattcgatctaatgg aaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatctatgattgcaa tactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaaccatcataaatcc cagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacacttacagtgca aaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacacaaccttttgt tacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggacaagtatggat agaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcagatggttcaat acgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatacgggaaacagt tgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgttcaagaatga tggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgatcggatccgag ccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatatggttaccatt attttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaaatagatggct taaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttatatcgaattcc tgcag 1855 <210> SEQ ID NO 120 <211> LENGTH: 12 <212> TYPE:PRT <213> ORGANISM: Mutant Preproricin linker region for Kallikrein,pap-292 <400> SEQUENCE: 120 Ser Leu Pro Arg Phe Lys Ile Ile Gly Gly PheAsn 1 5 10 <210> SEQ ID NO 121 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Neutrophil elastase linker region of pAP-294 <400> SEQUENCE:121 tctttgcttg gcattgctgt tcctggtaat tttaat 36 <210> SEQ ID NO 122 <211>LENGTH: 1855 <212> TYPE: DNA <213> ORGANISM: pAP-294 insert <400>SEQUENCE: 122 gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagtggcaacatgg 60 ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaacatattcccc 120 aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaagctacacaaac 180 tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagacatgatatacca 240 gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagttgaactctca 300 aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcatatgtggtcggc 360 taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaagatgcagaagca 420 atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttggtggtaattat 480 gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttgggaaatggtcca 540 ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactcagcttccaact 600 ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaagattccaatat 660 attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcaccagatcctagc 720 gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaagagtctaaccaa 780 ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaattcagtgtgtac 840 gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgcacctccacca 900 tcgtcacagt tttctttgct tggcattgct gttcctggta attttaatgctgatgtttgt 960 atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgttgatgttagg 1020 gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtctaatacagat 1080 gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatggaaagtgttta 1140 actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaatactgctgca 1200 actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcccagatctagt 1260 ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgcaaaccaacatt 1320 tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgttacaaccatt 1380 gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggatagaggactgt 1440 agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaatacgtcctcag 1500 caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagttgttaagatc 1560 ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatgatggaaccatt 1620 ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgagccttaaacaa 1680 atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccattattttgatag 1740 acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggcttaaataaaaa 1800 ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcctgcag 1855 <210> SEQ ID NO 123 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Mutant preproricin linker region for neutrophil elastase, pA<400> SEQUENCE: 123 Ser Leu Leu Gly Ile Ala Val Pro Gly Asn Phe Asn 1 510 <210> SEQ ID NO 124 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:Calpain linker region of pAP-296 <400> SEQUENCE: 124 tttttcaaaaatattgttac tcctagaacc ccccca 36 <210> SEQ ID NO 125 <211> LENGTH: 1855<212> TYPE: DNA <213> ORGANISM: pAP-296 insert <400> SEQUENCE: 125gaattcatga aaccgggagg aaatactatt gtaatatgga tgtatgcagt ggcaacatgg 60ctttgttttg gatccacctc agggtggtct ttcacattag aggataacaa catattcccc 120aaacaatacc caattataaa ctttaccaca gcgggtgcca ctgtgcaaag ctacacaaac 180tttatcagag ctgttcgcgg tcgtttaaca actggagctg atgtgagaca tgatatacca 240gtgttgccaa acagagttgg tttgcctata aaccaacggt ttattttagt tgaactctca 300aatcatgcag agctttctgt tacattagcg ctggatgtca ccaatgcata tgtggtcggc 360taccgtgctg gaaatagcgc atatttcttt catcctgaca atcaggaaga tgcagaagca 420atcactcatc ttttcactga tgttcaaaat cgatatacat tcgcctttgg tggtaattat 480gatagacttg aacaacttgc tggtaatctg agagaaaata tcgagttggg aaatggtcca 540ctagaggagg ctatctcagc gctttattat tacagtactg gtggcactca gcttccaact 600ctggctcgtt cctttataat ttgcatccaa atgatttcag aagcagcaag attccaatat 660attgagggag aaatgcgcac gagaattagg tacaaccgga gatctgcacc agatcctagc 720gtaattacac ttgagaatag ttgggggaga ctttccactg caattcaaga gtctaaccaa 780ggagcctttg ctagtccaat tcaactgcaa agacgtaatg gttccaaatt cagtgtgtac 840gatgtgagta tattaatccc tatcatagct ctcatggtgt atagatgcgc acctccacca 900tcgtcacagt tttttttcaa aaatattgtt actcctagaa cccccccagc tgatgtttgt 960atggatcctg agcccatagt gcgtatcgta ggtcgaaatg gtctatgtgt tgatgttagg 1020gatggaagat tccacaacgg aaacgcaata cagttgtggc catgcaagtc taatacagat 1080gcaaatcagc tctggacttt gaaaagagac aatactattc gatctaatgg aaagtgttta 1140actacttacg ggtacagtcc gggagtctat gtgatgatct atgattgcaa tactgctgca 1200actgatgcca cccgctggca aatatgggat aatggaacca tcataaatcc cagatctagt 1260ctagttttag cagcgacatc agggaacagt ggtaccacac ttacagtgca aaccaacatt 1320tatgccgtta gtcaaggttg gcttcctact aataatacac aaccttttgt tacaaccatt 1380gttgggctat atggtctgtg cttgcaagca aatagtggac aagtatggat agaggactgt 1440agcagtgaaa aggctgaaca acagtgggct ctttatgcag atggttcaat acgtcctcag 1500caaaaccgag ataattgcct tacaagtgat tctaatatac gggaaacagt tgttaagatc 1560ctctcttgtg gccctgcatc ctctggccaa cgatggatgt tcaagaatga tggaaccatt 1620ttaaatttgt atagtggatt ggtgttagat gtgaggcgat cggatccgag ccttaaacaa 1680atcattcttt accctctcca tggtgaccca aaccaaatat ggttaccatt attttgatag 1740acagattact ctcttgcagt gtgtgtgtcc tgccatgaaa atagatggct taaataaaaa 1800ggacattgta aattttgtaa ctgaaaggac agcaagttat atcgaattcc tgcag 1855 <210>SEQ ID NO 126 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Mutantpreproricin linker region for calpain, pAP-296 <400> SEQUENCE: 126 PhePhe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro 1 5 10 <210> SEQ ID NO 127<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Wild type linker region<400> SEQUENCE: 127 Ser Leu Leu Ile Arg Pro Val Val Pro Asn Phe Asn 1 510 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Oligonucleotide primers <400> SEQUENCE: 128 aattaaccct cactaaaggg 20<210> SEQ ID NO 129 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: T7primer <400> SEQUENCE: 129 gtaatacgac tcactatagg gc 22 <210> SEQ ID NO130 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Oligonucleotideprimer-Ricin-109 <400> SEQUENCE: 130 ggagatgaaa ccgggaggaa atactattgtaat 33 <210> SEQ ID NO 131 <211> LENGTH: 31 <212> TYPE: DNA <213>ORGANISM: Oligonucleotid primer-Ricin-99Eco <400> SEQUENCE: 131gcggaattcc gggaggaaat actattgtaa t 31 <210> SEQ ID NO 132 <211> LENGTH:18 <212> TYPE: DNA <213> ORGANISM: Oligonucleotide primer-Ricin267 <400>SEQUENCE: 132 acggtttatt ttagttga 18 <210> SEQ ID NO 133 <211> LENGTH:18 <212> TYPE: DNA <213> ORGANISM: Oligonucleotide primer-Ricin486 <400>SEQUENCE: 133 acttgctggt aatctgag 18 <210> SEQ ID NO 134 <211> LENGTH:18 <212> TYPE: DNA <213> ORGANISM: Oligonucleotide primer-Ricin725 <400>SEQUENCE: 134 agaatagttg ggggagac 18 <210> SEQ ID NO 135 <211> LENGTH:18 <212> TYPE: DNA <213> ORGANISM: Oligonucleotide primer-Ricin 937<400> SEQUENCE: 135 aatgctgatg tttgtatg 18 <210> SEQ ID NO 136 <211>LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Oligonucleotide primer-Ricin1151 <400> SEQUENCE: 136 cgggagtcta tgtgatga 18 <210> SEQ ID NO 137<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Oligonucleotideprimer-Ricin 1399 <400> SEQUENCE: 137 gcaaatagtg gacaagta 18 <210> SEQID NO 138 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:Oligonucleotide primer-Ricin 1627 <400> SEQUENCE: 138 ggattggtgttagatgtg 18 <210> SEQ ID NO 139 <211> LENGTH: 19 <212> TYPE: DNA <213>ORGANISM: Oligonucleotide primer-Ricin 1729C <400> SEQUENCE: 139ataacttgct gtcctttca 19 <210> SEQ ID NO 140 <211> LENGTH: 27 <212> TYPE:DNA <213> ORGANISM: Oligonucleotide primer-Ricin 1729C Xba <400>SEQUENCE: 140 cgctctagat aacttgctgt cctttca 27 <210> SEQ ID NO 141 <211>LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Ricin109-Eco Oligonucleotide<400> SEQUENCE: 141 ggaggaatcc ggagatgaaa ccgggaggaa atactattgt aat 43<210> SEQ ID NO 142 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Ricin1729-PstI <400> SEQUENCE: 142 gtaggcgctg cagataactt gctgtccttt cag33

I claim:
 1. A purified and isolated nucleic acid having a nucleotidesequence encoding an A chain of a ricin-like toxin, a B chain of aricin-like toxin and a heterologous linker amino acid sequence linkingthe A and B chains, the heterologous linker sequence containing acleavage recognition site for a cancer-associated protease, wherein thecancer-associated protease is selected from the group consisting of:cathepsin B, a matrix metalloproteinase, cathepsin L, cathepsin D,urokinase-type plasminogen activator, tissue-type plasminogen activator,human prostate-specific antigen, kallikrein, neutrophil elastase, andcalpain.
 2. The nucleic acid sequence of claim 1, wherein the A chainis: ricin A chain, abrin toxin A chain, diphtheria toxin A chain, orDomain II/III of Pseudomonas exotoxin.
 3. The nucleic acid sequence ofclaim 1, wherein the B chain is: ricin B chain, abrin toxin B chain,diphtheria toxin B chain, or Domain I of Pseudomonas exotoxin.
 4. Thenucleic acid sequence of claim 1, having the nucleotide sequenceaccording to SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ IDNO:11; SEQ ID NO:86; SEQ ID NO:89; SEQ ID NO:92; SEQ ID NO:95; SEQ IDNO:98; SEQ ID NO:101; SEQ ID NO:104; SEQ ID NO:107; SEQ ID NO:110; SEQID NO:113; SEQ ID NO:116; SEQ ID NO:119; SEQ ID NO:122; or SEQ IDNO:125.
 5. A plasmid incorporating the nucleic acid of claim
 1. 6. Aplasmid incorporating the nucleic acid of claim
 4. 7. A baculovirustransfer vector incorporating the nucleic acid of claim
 1. 8. Abaculovirus transfer vector incorporating the nucleic acid of claim 4.9. A purified and isolated nucleic acid having a nucleotide sequenceencoding an A chain of a ricin-like toxin, a B chain of a ricin-liketoxin and a heterologous linker amino acid sequence linking the A and Bchains, the heterologous linker sequence containing a cleavagerecognition site for a Plasmondium falciparum protease.
 10. The nucleicacid sequence of claim 9, wherein the A chain is: ricin A chain, abrintoxin A chain, diphtheria toxin A chain, or Domain II/III of Pseudomonasexotoxin.
 11. The nucleic acid sequence of claim 9, wherein the B chainis: ricin B chain, abrin toxin B chain, diphtheria toxin B chain, orDomain I of Pseudomonas exotoxin.
 12. The nucleic acid sequence of claim9, having the nucleotide sequence according to SEQ ID NO:13; SEQ IDNO:15; SEQ ID NO:17; SEQ ID NO:19; or SEQ ID NO:21.
 13. A plasmidincorporating the nucleic acid of claim
 9. 14. A plasmid incorporatingthe nucleic acid of claim
 12. 15. A baculovirus transfer vectorincorporating the nucleic acid of claim
 9. 16. A baculovirus transfervector incorporating the nucleic acid of claim
 12. 17. A purified andisolated nucleic acid having a nucleotide sequence encoding an A chainof a ricin-like toxin, a B chain of a ricin-like toxin and aheterologous linker amino acid sequence linking the A and B chains, theheterologous linker sequence containing a cleavage recognition site fora viral protease, wherein the viral protease is selected from the groupconsisting of: human cytomegalovirus, human herpes virus, varicellazoster virus, hepatitis A virus, hepatitis C virus, Epstein-Barrvirus-specific protease, and infectious laryngotracheitis virus.
 18. Thenucleic acid sequence of claim 17, wherein the A chain is: ricin Achain, abrin toxin A chain, diphtheria toxin A chain, or Domain II/IIIof Pseudomonas exotoxin.
 19. The nucleic acid sequence of claim 17,wherein the B chain is: ricin B chain, abrin toxin B chain, diphtheriatoxin B chain, or Domain I of Pseudomonas exotoxin.
 20. The nucleic acidsequence of claim 17, having the nucleotide sequence according to SEQ IDNO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:48; SEQ IDNO:52; SEQ ID NO:74; SEQ ID NO:77; SEQ ID NO:80; or SEQ ID NO:83.
 21. Aplasmid incorporating the nucleic acid of claim
 17. 22. A plasmidincorporating the nucleic acid of claim
 20. 23. A baculovirus transfervector incorporating the nucleic acid of claim
 17. 24. A baculovirustransfer vector incorporating the nucleic acid of claim
 20. 25. Apurified and isolated nucleic acid having a nucleotide sequence encodingan A chain of a ricin-like toxin, a B chain of a ricin-like toxin and aheterologous linker amino acid sequence linking the A and B chains, theheterologous linker sequence containing a cleavage recognition site fora Candida acid protease.
 26. The nucleic acid sequence of claim 25,wherein the A chain is: ricin A chain, abrin toxin A chain, diphtheriatoxin A chain, or Domain II/III of Pseudomonas exotoxin.
 27. The nucleicacid sequence of claim 25, wherein the B chain is: ricin B chain, abrintoxin B chain, diphtheria toxin B chain, or Domain I of Pseudomonasexotoxin.
 28. The nucleic acid sequence of claim 25, having thenucleotide sequence according to SEQ ID NO:50 or SEQ ID NO:54.
 29. Aplasmid incorporating the nucleic acid of claim
 25. 30. A plasmidincorporating the nucleic acid of claim
 28. 31. A baculovirus transfervector incorporating the nucleic acid of claim
 25. 32. A baculovirustransfer vector incorporating the nucleic acid of claim
 28. 33. Thenucleic acid of claim 1, wherein the A chain is ricin A chain, the Bchain is ricin B chain, and the heterologous linker contains a cleavagerecognition site for matrix metalloproteinase.
 34. The nucleic acid ofclaim 33, wherein the heterologous linker contains a cleavagerecognition site for matrix metalloproteinase-9.
 35. The nucleic acid ofclaim 1, wherein the A chain is ricin A chain, the B chain is ricin Bchain, and the heterologous linker contains a cleavage recognition sitefor human prostate-specific antigen.
 36. The nucleic acid of claim 17,wherein the A chain is ricin A chain, the B chain is ricin B chain, andthe heterologous linker contains a cleavage recognition site epatitis Cvirus.